Miniaturized Digestion and Extraction of Surface Proteins from ...

Research ArticleMiniaturized Digestion and Extraction of SurfaceProteins from Candida albicans following Treatment withHistatin 5 for Mass Spectrometry Analysis

Shirley Fan1 Eduardo B Moffa23 Yizhi Xiao23

Walter L Siqueira23 and Ken K-C Yeung12

1Department of Chemistry Faculty of Science University of Western Ontario London ON Canada N6A 5B72Department of Biochemistry Schulich School of Medicine and Dentistry University of Western OntarioLondon ON Canada N6A 5C13Schulich Dentistry Schulich School of Medicine and Dentistry University of Western OntarioLondon ON Canada N6A 5C1

Correspondence should be addressed to Ken K-C Yeung kyeunguwoca

Received 22 June 2016 Revised 25 October 2016 Accepted 31 October 2016

Academic Editor Christian Huck

Copyright copy 2016 Shirley Fan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A common approach to isolate surface proteins from fungal and bacterial cells is to perform a proteolytic cleavage of proteins on thesurface of intact cells suspended in solution This paper describes miniaturization of this technique in which cells are adhered onglass surfaces and all sample treatments are conducted at 120583L volumes Specifically Candida albicans cells were attached onto HSA-coated glass slides By depositing the appropriate reagent solutions on the adhered cells we successfully performed cell washingtreatment with antifugal peptide Histatin 5 and a proteolysis on intact cells with trypsinThe resulting peptides were subsequentlyanalysed by mass spectrometry In general the data obtained was similar to that collected with suspended cells in much largersample volumes However our miniaturized workflow offers the benefit of greatly reducing the consumption of cells and reagents

1 Introduction

Candida albicans is a very common fungus found within thegenitourinary and gastrointestinal tracts as well as on thesurface of skins [1] It exists within humans in a commensalrelationship where it benefits without causing damage [2]However individuals with compromised immune systemscan develop the infection known as candidiasis [2 3]The most commonly used method to treat infections isto administer antifungal drugs orally or systematically intothe circulatory system [2 4] Despite these mechanisms Calbicans has developed resistance towards them [4] and thusthe discoveries of new therapeutic approaches are needed

For infections to begin adhesion to oral surfaces ofprotein films is the initial step [5 6] From these proteinssalivary proteins such as histatins statherins and acidicproline-rich ones have exhibited antifungal properties [7 8]The family known as histatins (HTN) comprised of HTN1

HTN3 and HTN5 have been of increasing interest due to itsclear ability to control and kill C albicans [5 7 9 10] Studiestargeting the effects of HTN5 on cellular respiration at themitochondrial level have been performed however the exactmechanism remains unknown [9 11 12] Mass spectrometry-(MS-) based proteomics is therefore the next logical choice ofmethod in studying C albicans and its influence by HTN5

MS is becoming an increasingly popular tool in studyingmicroorganisms Common applications include protein pro-filing for taxonomy classification as well as clinical differen-tiation between pathogenic and nonpathogenic species [13ndash17] The degree of classification achieved has been reporteddown to the genus species and strain levels [18] Massspectral protein profiling has also been incorporated intostudying the response of bacterial cells to drug treatment [19]Typically the analysis of the total proteome of lysed cells ispreferred for a comprehensive nontargeted characterizationBut the use of total cell lysates has its drawbacks Due to the

Hindawi Publishing CorporationInternational Journal of ProteomicsVolume 2016 Article ID 9812829 12 pageshttpdxdoiorg10115520169812829

2 International Journal of Proteomics

complexity of the proteome and background of lysed cellssample cleanup andor fractionation is crucial to obtaininghigh qualityMSdata [16]The identification of a large numberof proteins also requires a substantial effort in data analysisAlternatively the analysis of intact whole cells offers thebenefit of reduced complexity Anhalt and Fenselau wereamong the earliest to report the analysis of whole cellsfor species identification [20] Through gentle heating inthe ion source phospholipids were vaporized leading tothe production of characteristic mass spectra Alternativelyspecies identification through proteins and peptides fromwhole cell analyses has also been reported [21ndash23]

When applying MS to study changes in protein levels ofC albicans upon treatment with HTN5 the global approachof studying the total proteome was certainly appropriateHowever our group previously observed a reduction incell adhesion property upon administration of HTN5 in90 minutes suggesting an effect of HTN5 on the surfaceproteins [24] Hence the targeted approach on the surfaceproteins is logical for studying the influence of HTN5 In2006 Rodrıguez-Ortega et al reported a protocol calledldquocell shavingrdquo which allowed them to focus on proteinspartially exposed on the outside of cells [25] This was doneby subjecting whole live cells to proteolytic enzymes in asolution The surface peptides cleaved from intact cells weresubsequently isolated for MS analyses Others have alsoused a similar approach towards studying surface proteinsfrom other fungal and bacterial species [26 27] Despite itseffectiveness the procedure of ldquocell shavingrdquo is still generallytime-consuming Cells grown in media were harvested andwashed followed by overnight tryptic digestion for 18ndash20hours in mL volume solution The sample mixture was thencentrifuged and the supernatant was collected for fraction-ation andor enrichment prior to injection into the liquidchromatography-mass spectrometer [28ndash30] Furthermoreto overcome biological variation and growth variability theentire procedure was typically repeated for multiple timesthus requiring large quantities of cells and reagents

In this study the aim is to refine and miniaturize theexisting cell shaving technique by performing the procedureson adhered C albicans cells rather than suspended cells insolution We will demonstrate that the attachment of cells onsurfaces allows easy introduction and removal of 120583L volumereagents solutions with a micropipette Furthermore treat-ment with antifungal peptide HTN5 is readily performedon the adhered cells We will also demonstrate that the 120583Lsample volume resulting from our miniaturized approachis directly compatible with subsequent MALDI MS andorLC ESI MSMS analyses Importantly the resulting MS datashould be comparable to previous information obtained withthe conventional in solution approach that required muchlarger quantities of reagent and cells

2 Materials and Methods

21 C albicans Culturing The strain of C albicans used forall experiments was ATCC90028 Stock cultures of cells weregrown on plates containing Sabouraud dextrose agar (SDA)medium (Becton Dickson and Company NJ USA) The

(a)

(b)

(c)

Figure 1 Schematics of the miniaturized procedures of cell adhe-sion treatment and tryptic digestion Description is presented inMaterials and Methods

inoculated plates were incubated at 37∘C for 48 hours A thirdof a loop of the colonies were transferred into 45mL of yeastnitrogen base (YNB) supplemented with 50mM sucrose(Sigma Aldrich MO USA) The mixture was then incubatedat 37∘C for 21 hours under shaking at 200 rpm Following theincubation the mixture was centrifuged at 6000 rpm for 5minutes The pellet was resuspended in phosphate bufferedsaline solution (PBS pH 74 Sigma Aldrich) The final cellsuspension was stored at 4∘C until use

22 Adhesion of C albicans to Glass Slides and TreatmentwithHTN5 ADakodelimiting pen composed of 60ndash100 1-bromopropane and 5ndash10 dipentene (Cedarlane BurlingtonCA) was used to draw circles with an inner diameter ofroughly 6mm on standard microscope glass slides (Fig-ure 1(a)) The hydrophobic ink functioned as a barrier tosurround a droplet of aqueous sample solution A total ofthree circles were made on each glass slide (Figure 1(b))During an experiment each sample spot was covered with aninverted glass vial containing a wet cotton which maintaineda high humidity environment and minimized evaporation ofthe sample droplet (Figure 1(c)) To prepare for cell adhesionthe glass surface within the hydrophobic circles was treatedwith 25 120583L of 010mgmL human serum albumin (HSA)solution (Sigma Aldrich) for 2 hours at room temperature(RT) based on a protocol previously reported by our group[5] The HSA-coated surface was then washed three timeswith water Next 25120583L of C albicans suspension at aconcentration of 107 cellsmL was introduced to the HSA-coated slideThe adhesion was carried out under a 90-minute

International Journal of Proteomics 3

incubation at RT The surface with adhered cells was thenrinsed three times with PBS to remove the unattached cellsFor theHTN5 treatment 25120583L ofHTN5 solution (ChinaPep-tides Company Shanghai China) at various concentrationswas deposited to the cells When not specified a HTN5concentration of 30120583gmL was used The HTN5 treatmentwas done for 90min at RT which was the minimum time toobserve a clear effect of HTN5 on C albicans [24] The finalsamples of treated cells on the glass slide were washed threetimes with water

23 Tryptic Digestions on Solid Support In a previous reportthe trypsin digestion of surface proteins on C albicanscells suspended in buffer solution was performed at atrypsin concentration of 10 120583gmL trypsin for up to 20min[28] The authors also performed propidium iodide stainingexperiment to confirm that the C albicans cells were notpermeabilized by trypsin In this work digestions wereperformed on adhered C albicans cells with or withoutHTN5 treatment on glass surfaces within the hydropho-bic circles at RT It was therefore necessary to reoptimizethe digestion time and trypsin concentration Due to thelarge number of experiments required to map these twoparameters we decided to first evaluate the digestion usinga standard protein cytochrome c from horse heart (Cata-logue number C2506 Sigma Aldrich) For all digestions inthis work tosyl phenylalanyl chloromethyl ketone- (TPCK-)treated trypsin resuspended in 50mM ammonium bicarbon-ate (Sigma Aldrich) was used

To determine the optimal digestion time for cytochromec a very high concentration of trypsin was initially usedto ensure that it was not a limiting factor Specifically036 120583L of 01mgmL cytochrome c was mixed with 036120583Lof 10mgmL trypsin to perform the digestion directly on aMALDI sample target plate under a range of digestion timesfrom 025 to 60min After the optimal time was determinedbased on MALDI MS results which turned out to be 10minthe digestion was then performed on adhered C albicanscells The trypsin concentration used was reduced to a rangecomparable to that reported in the literature [28] namely 05to 20120583gmL and a volume of 3 120583L was used The optimaltrypsin concentration was determined to be 15 120583gmL basedon MALDI MS results Finally using this trypsin concentra-tion we reexamined the digestion time study with adheredC albicans cells The MALDI MS results confirmed that theoptimal digestion time remained at 10min Finally followingthe digestion step performed on C albicans cells the trypsinsolution now with resulting peptides was recovered using amicropipette for subsequent MALDI MS or LC ESI MSMSanalyses

24 MALDI MS Analysis The 120572-cyano-4-hydroxycinnamicacid (CHCA) matrix solution was prepared at 55mgmL in6mM ammonium phosphate monobasic 50 acetonitrileand 01 trifluoroacetic acid (Sigma Aldrich) The recoveredpeptides 072120583L from cytochrome c or 3 120583L fromC albicanswere mixed in a 1 1 (vv) ratio with this CHCA matrix solu-tion All sample-matrix mixtures were spotted at a volumeof 075 120583L on a 384 well Opti-TOF 123 times 83mm SS MALDI

plate (Sciex MA USA) The instrument is equipped with a349 nm OptiBeam On-Axis laser with a pulse rate at 400HzData acquisition and processing were done using TOF-TOFSeries Explorer (Sciex) and Data Explorer The spectra wereacquired in Reflectron positive mode from 500ndash3500 mzPeak lists were created using the following parameters apeak density of 10 per 25Da minimal signal-to-noise (SN)of 10 minimum area of 50 and a maximum peak per spotof 200 Second fragmentation MSMS was also performedvia postsource decay (PSD) using the 1 kV in positive ionacquisition mode

25 Nano-HPLC ESI MSMS Prior to injection for LCMS the peptide sample (3 120583L) underwent sample cleanupusing C18 ZipTip (Millipore MA USA) The eluted peptideswere then dried and concentrated by the Vacufuge vacuumconcentrator (Eppendorf Germany) This was performed atRT for 10minutes at 14000 rpmOnce dried the peptideswereresuspended in 10 120583L of 01 formic acid (Sigma Aldrich)with 8 120583L of this sample injected to LC MS

Nano-HPLC was carried out on an Thermo ScientificEasy nLC II instrument (Thermo Scientific CA USA) The85-minute gradient composition ranged from 5 to 55 ofsolvent B which was 975 acetonitrile and 01 formic acid(Sigma Aldrich) Solvent A is 01 FA in water The flow rateused was 200 nLmin at a pressure of 280 bar The volumeof sample injected was 8 120583L ESI was conducted using avoltage of 20 kV with an ion transfer capillary temperatureof 2500∘C

MS analyses were performed on a Thermo Scientificlinear trap quadrupole (LTQ) Velos ion mass spectrom-eter (Thermo Scientific) Positive mode data acquisitionsand processing were done using Thermo Xcalibur 210SP11162 software (Thermo Scientific USA) The MS scanmz range used was 0ndash2000 MSMS was done via collisioninduced dissociation (CID) using helium (He) as the inertgas

26Database Searches AllMALDIMSspectrawere searchedagainst the Uniprot protein database (SwissProt 2015 02547 599 entries) on MASCOT provided by Matrix Science(httpwwwmatrixsciencecom) The search parametersused forC albicanswere (1) trypsinwith onemissed cleavage(2) fungi as the taxonomy (3) a variable modification forthe oxidation of methionine and (4) a mass tolerance ofplusmn70 ppm

All ESI MSMS spectra were run through the ThermoProteome Discoverer version 130339 software (ThermoFisher Scientific Inc USA) using the algorithm known asSEQUEST The C albicans database chosen to search resultsagainst was the UniProt Knowledgebase more commonlyknown as UniProtKB (UniProt Consortium httpwwwuniprotorg)Thedata analysis programSEQUESTwas usedfor protein identification The search parameters were asfollows (1) enzymatic cleavage by trypsin with up to 2missedcleavages (2) signal-to-noise ratio (SN) of 15 (3) precursormass tolerance of plusmn2Da and (4) fragment mass tolerance ofplusmn08Da


2 120583m

(a) Control

2 120583m

(b) 30120583gmL

2 120583m

(c) 25120583gmL

2 120583m

(d) 20120583gmL

Figure 2 Microscope images of C albicans adhered on slides after a 2-hour treatment with Histatin 5 The concentrations of HTN5 areindicated in (bndashd) with units of 120583gmL The average cell counts (119899 = 4) with standard deviations were (a) 110 plusmn 8 (b) 16 plusmn 8 (c) 25 plusmn 5 and(d) 42 plusmn 4

3 Results and Discussion

31 C albicans Cell Adhesion and HTN5 Treatment Our re-search team previously studied and reported fungicidal activ-ity of HTN5 on C albicans [24] The results indicated thatpreexposition of HTN5 to oral epithelial cells diminishedthe adhesion of C albicans to the epithelium Since theminiaturized assay in this work is performed on adheredcells it is important to determine an optimal dosage ofHTN5which is sufficient to cause detected changes in proteinabundance but not too high to eradicate and desorb all cellsfrom the glass surface

To begin we reproduced the adhesion of C albicans onHSA-coated glass surfaces as previously reported [5] andthen the cells were treated with varying concentrations ofHTN5 Given the physiological concentration ofHTN5 in theoral cavity 10ndash30 120583M[31] we performedHTN5 treatments at20 25 and 30 120583gmL (Figures 2(b) and 2(c)) A 2-hour treat-ment time was selected based on the previous observationthat 90min was sufficient to cause a reduction in C albicanscolonization [24] Figure 2 shows the typical microscopicalviews of the adhered cells after HTN5 treatments Theaverage cell count numbers presented in the caption referto counts within the microscopical views As expected areduction in cell counts after exposure to HTN5 was evidentand the effect was greater at higher HTN5 concentration

Given that sufficient cells remained adhered even at thehighest concentration studied 30 120583gmL we chose to use thisconcentration in subsequent HTN5 treatments to maximizechanges in the C albicans protein levels

32 Tryptic Digestions Performed on Adhered Cells Prior toperforming digestion of C albicans cells adhered on glasssurfaces an optimization of the tryptic digestion conditionswas conducted The first objective was to determine theshortest reaction time require to complete a digestion withan excess quantity of trypsin We chose to conduct thisstudy on a standard protein cytochrome c In contrast toworking with cells the use of a standard protein allows us toeasily control the amount of starting materials and providesa set of well-defined tryptic peptide products as the reactionend-point Triplicate digestions at RT were conducted underthe concentrations of 005mgmL for cytochrome c and05mgmL for trypsin at a total volume of less than 1 120583LThereaction time durations studied were 025 05 075 1 2 3 45 10 15 20 25 30 and 60 minutes It was observed that theshorter digestion times of 15 seconds to 4 minutes producedconsiderably low peptide signals (data not shown) The mostcritical changes in number of peptides andor peak intensitiesoccurred between 5 and 15 minutes of digestion time A totalof eight tryptic peaks of cytochrome c with up to 1 missedcleavage were detected (119898119911 of 63439 116862 126058


129672 145481 149570 159878 and 163382) The changesin peak intensities of these eight peptides between digestionperiods of 5 to 15 minutes are shown in Figure S1 (seeSupporting Information in Supplementary Material availableonline at httpdxdoiorg10115520169812829) Essentiallyhighest signals were recorded from the digestion time of 10minutes Beyond 10 minutes the digestion appeared to becompleted and the opposing effect of peptide loss due tononspecific adsorption on surfaces was speculated

Using the optimal digestion time of 10 minutes forcytochrome c we subsequently optimized the trypsin con-centration for the adhered C albicans cells The trypsinconcentrations used for this experiment were 05 075 115 and 2120583gmL Five replicate digestion experiments wereconducted by depositing 3 120583L of trypsin solution at variousconcentrations on adhered cells as shown in Figure 1 Fromthe 25 resulting mass spectra peaks with SN above 10were extracted for their values of 119898119911 and peak heightBackground signals from trypsin and HSA were removedfrom the list and averages of peak intensities were takenfrom replicates The two lowest concentrations studied 05and 075 120583gmL resulted in noticeably fewer peaks andso only the results from the higher three concentrationswere shown in Table S1 (under Supporting Information)Based on the number of signals observed our results indi-cated that 15 120583gmL was the optimal In addition the peakintensities were also highest at this concentration in mostcases

Once again the optimal digestion time of 10min waspreviously determined using cytochrome c In case thisoptimal value is substrate-specific we reexamined the effectof digestion time for C albicans at a trypsin concentrationof 15 120583gmL Four digestion times 5 10 15 and 20 minutesin replicates of four were studied The results are presentedin Table S2 which confirmed that 10 minutes remainedto be optimal in yielding the highest number of signalsPrevious work performed the isolation of surface peptidesfrom C albicans in solution under digestion times from fiveto twenty minutes [28] They reported that five minutes wassufficient for the release of easily accessible surface peptidesand as the time was increased the number of resultingpeptides increased as well Our data generally agreed withtheir observations Even though the greatest number of peakswas observed at 10 minutes the peak intensities of somepeaks continued to increase beyond 10 minutes for example119898119911 of 6560 8514 11115 and 24470 However we havenot yet confirmed which of these signals originated fromC albicans and thus the comparison should not be basedon the intensities of selected peaks This led us back to theconclusion of 10 minutes being optimal based on the highestnumber of observed peaks

Most importantly the results demonstrated the successfultryptic digestion performed on C albicans adhered on glasswith as little as 3 120583L of trypsin solution Using the establishedoptimal reaction time of 10min and trypsin concentration of15 120583gmL digestions were performed finally on C albicanscells treated with HTN5 The resulting peptides were recov-ered for analysis by LC ESI MSMS for identification

33 Analyses of Peptides from C albicans by Nano-HPLCESI MSMS ESI MS and MSMS data were generated forsamples collected from the tryptic digestion of C albicanscells with and without HTN5 treatments The entire studywas performed in replicates of six for each of the two groupsand the results were searched against the C albicans databasefor protein identification It is noteworthy that the natureof our miniaturized assay while reducing the consumptionof reagents and cells also produces a limited quantity ofpeptides for protein identification It was estimated that lessthan 2 120583g of materials was injected for each LC MS analysisFor this reason we have taken a less stringent approachin setting our database search parameters and so we caninclude results with moderate to high levels of confidenceFurthermore instead of focusing only on peptides withsubstantial changes in abundance after exposure to HTN5we also take the inclusive approach and present the differentlevels of abundance changes resulting from the treatmentTo help readers distinguishing the peptides detected withincreased intensities from those with reduced intensitiesfollowing HTN5 treatment the results are divided into twotables Table 1 presents the precursor ions with averageintensities that were higher from the HTN5 treated samplescompared to the control whereas Table 2 shows the ionswith lower intensities from the treated group compared tothe control The 119901 values resulting from 119905-test are included inthe tables to illustrate the significance of the signal intensitychanges Entries with high119901 values (gt01) should be treated asstatistically indistinguishable The signal intensities of theseions were highly variable within the replicate measurementswhich resulted in large standard deviations and low statisticalconfidences

The confidences of protein assignments were also illus-trated in the tables as 119883Corr values 119883Corr values above 2 areusually indicative of a good correlation It is noteworthy thatsome observed precursor ions were matched with multipletheoretical precursor ions for example observed119898119911 129759was matched with theoretical 119898119911 129854 129749 129769129770 129775 and 129867 Readers should take into accountthe 119883Corr scores when interpreting the results as some ofthese protein assignments are only putative Finally theprotein entries were sorted according to their biologicalfunctions but these listed functions were taken straight outof the Uniprot database based on the accession numbersresulting from the search (listed next to the protein names inTables 1 and 2) Further experimental verifications were notconducted in this work

In the previous study of peptides resulting from cellshaving of C albicans in solution [28] the authors reportedthe detection of proteins with the following functionsmetabolism cell defense and virulence transport and pro-tein fate Likewise the profiling ofC albicans surface proteinswas reported by two other groups in different analyticalapproaches [29 32] Broadly speaking similar functions wereobserved in Tables 1 and 2 While these previous reportsoffered more comprehensive listings of proteins with cellwall functions our work focused on the changes in surfaceproteins upon HTN5 treatment

6 International Journal of ProteomicsTa

ble1Proteins

identifi

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mam

iniaturiz

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decreasedMSsig

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replicatesProteinsind

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Theoretic

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Aminoacid

sequ

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Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

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Virulence

128483

128665

2700

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Transcrip

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Cand

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-7(A

0A0A

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129587

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tAVtAT

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(A0A

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TSIPPA

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TtLttIS

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tTttT

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(A0A

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Mito

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drial-

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117588

117515

6400

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3900

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1100lt000

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(A0A

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1296

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(Q92211)

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1500

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3200plusmn

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mGmALF

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Clusteredmito

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DNAbind

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80477

80444

4200

0plusmn

2200

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3100

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KQPA

VFSRI

DNApo

lymerase

(Q5A

4E5)

1256

128483

128353

2700

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2800

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(A0A

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1267

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333142

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1400lt000

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International Journal of Proteomics 7Ta

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tinued

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nal

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Charges119883

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Con

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Y81)

3303

465996

466019

1500

0plusmn

1100

3200plusmn

2700

000

014

RRS

VSYS

PGPsSIKS

QLP

HLT

SSST

TtssVQ

SPP

PPPP

SQPP

RG

361

417360

417373

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

KtV

TsIN

GSP

PPLE

tAPsSH

HNVPIDFIHFK

KESD

RT

Uncharacterized

protein

(A0A

0A3E

PD4)

3312

436859

436789

3300

0plusmn

2100

07100plusmn

1300

0020

KNMQFP

PYQVS

sHNsSEtSQ

sIPN

TPSITR

QVE

SNTR

S

Transcrip

tionalregulatory

proteinLE

U3

(A0A

0A3B

TY8)

3354

436790

KtPTT

TTTT

TTtANGNTS

NGNTS

NGNsTGKT

ATA

ATAT

KSNtK

Transcrip

tionproteinfamilylowast

(A0A

0A3C

3Y1)

319

Protein

synthesis

80477

80541

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQTS

LNDKC

Manno

syltransfe

rase

familylowast

(A0A

0A3C

UB4

)1

254

80445

RVKI

DSD

KS

ProteintransporterS

EC24

(A0A

0A3E

HG4)

151

117588

117398

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KTK

QFN

DsK

KK

Vesic

letetheringprotein

familylowast

(A0A

0A6K

IG6)

1201

128483

128372

2700

0plusmn

9500

1400

0plusmn

2800

0016

KtAGsN

HNsEKK

Ribo

somalRN

A-processin

gprotein12

(A0A

0A4B

GJ7)

1251

412103

411993

3100

0plusmn

2300

01700

0plusmn

7200

013

KFF

sDNIANDLA

tTTT

TTTT

TNTG

AtSV

HPIL

QVDAIK

YCA

SCS

Eproteinfamilylowast

(A0A

0A6L

8P2)

3324

436859

437093

3300

0plusmn

2100

07100plusmn

1300

0020

KES

SSTA

DQPS

VVPP

QES

HKD

TVET

PKPE

VtEtsV

EAtKE

Transla

tioninitiationfactor

4G(A

0A0A

4CDK4

)3

299

436703

KNPT

PTPT

PTPT

PTPN

NLA

QGVDsSST

LDV

EtTL

tGLtRR

I

Imidazoleglycerolpho

sphate

synthase

cycla

sesubu

nit

(A0A

0A6I238)

321


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Sign

allin

g

350340

350460

4300

0plusmn

8300

3100

0plusmn

2500

0013

KRS

SITtPtPP

LTTT

HSSNGNGNGNV

NVNVNsK

R

Peroxisometransmem

brane

receptorlowast

(A0A

0A3Z

WC2

)3

330

411551

411676

3100

0plusmn

2000

01700

0plusmn

7200

0017

KQST

TNTsTL

ssTtAAST

LATS

NNTQ

PDTY

TSTS

TSIRG

Pleckstrin

homologydo

mainlowast

(A0A

0A6L

5L9)

3333

441651

441487

2900

0plusmn

2700

01600

0plusmn

1100

010

RQHPD

PLSN

QsN

FNsN

TINNYS

NYR

SsTRS

GLD

PsQRH

RhoGTP

asea

ctivating

proteindo

mainlowast

(A0A

0A3B

MU5)

3412

465996

466032

1500

0plusmn

1100

3200plusmn

2700

000

014

KDLP

IGYILH

mIN

LcPN

IVsLNLG

NLSLsTD

YEISRS

TIHKY

F-bo

xproteinCO

S111

(A0A

0A3C

YY6)

3368

465821

KWNKE

KIEL

DsPLIVS

YVSSLC

NGGGGGIIT

NsTNST

ttNSK

Pentatric

opeptid

erepeat

(PPR

)protein

familylowast

(A0A

0A3C

GE7

)111

Misc

ellaneou

s

128483

128542

2700

0plusmn

9500

1400

0plusmn

2800

0016

RNDsD

tsLsK

EInsulin

indu

cedprotein

familylowast

(A0A

0A3C

RM0)

116

6RYtmNEV

FKV

GYF

domainlowast

(A0A

0A3C

0N8)

333128

333159

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

ETsQ

FMGNAES

EDLtGNGLL

TSTL

AVLS

SIS

Orfin

Calbicans

major

repeat

sequ

enceR

B2region

(Q5A

475)

3315

333128

333244

6300

0plusmn

1200

1400

0plusmn

1400

023

KLS

SLGNHGTtTT

SSLS

SSsSsSIsNNT

SIAKI

CBSdo

mainlowast

(A0A

0A6JTB

4)3

346

337660

337659

4300

0plusmn

1200

03300

0plusmn

2800

0RKQ

QDQNEV

AGAAAAT

TTTA

tAtAT

AAT

NWKP

KN

321

349815

349857

3400

0plusmn

8900

1800

0plusmn

1600

00054

KSSsLIK

NsTsSNQSsPA

TSTN

TSIV

DVPIEK

SWDrepeatproteinlowast

(A0A

0A3C

A48)

3321

417360

417295

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

RIN

NNNDKs

sILsNITTT

NTT

TGTG

TTNTtT

VPS

IKTK

R(A

0A0A

6IY5

3)424

349815

349948

3400

0plusmn

8900

1800

0plusmn

1600

00054

KAFS

SsKL

TSDSA

NStNStNsTSM

SILG

NDKD

CDKinhibitorP

HO81

(A0A

0A6M

Y31)

3316

389536

389486

3600

0plusmn

4100

2600

0plusmn

3100

00037

RISRP

NGVG

GISTS

GSSSP

TTEF

VTP

QAs

KsSV

DQNKK

RUbiqu

itininteractingmotiflowast

(C4Y

NW3)

3329

442103

442109

3200

0plusmn

1900

01700

0plusmn

7200

0080

RHLILG

YKItV

VtDHQSLTsVMTS

SSRP

ENNRM

IRW

Aspartic

peptidasefam

ilylowast

(A0A

0A4C

VF8

)3

383

393914

394016

3600

0plusmn

2400

01700

0plusmn

1800

000026

RNVNGSG

StNtN

TMtRLD

sTTIASSLF

CRQLY

FNLL

SKD

Globinfamilylowast

(A0A

0A4C

D75)

3311

437257

437390

9600plusmn

4600

5400plusmn

1800

0058

RNNsT

VSST

NSL

mSN

NsD

TNtAAT

AAT

AAT

SGS

TTNNVKR

M(A

0A0A

3XE4

6)338


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

441615

441482

2900

0plusmn

2700

01600

0plusmn

1100

010

RNGtYSSsStSSSsTSSVS

SSSA

TTANGES

LNST

THNIQ

LERT

Sec2plowast

(A0A

0A6JGN5)

3289

465996

466133

1500

0plusmn

1100

3200plusmn

2700

000

014

KQNYV

DPG

QIAIKGLK

GFV

NLG

AtCF

MssILQtLIH

NPL

IKY

Ubiqu

itincarboxyl-te

rminal

hydrolase

(C4Y

KS7)

3369

466093

KYD

KPmED

TEEIDDVtSISK

sINEQ

IDDPF

sQFN

SVTL

RY

Protein-serin

ethreon

ine

kinase

(A0A

0A4A

NB2

)35

1

Uncharacterized

117588

117373

6400

0plusmn

4000

2200

0plusmn

1100lt000

001

KtIm

tItIK

I(Q

59T3

9)1

207

333128

333260

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

RIsNIITL

NSSLS

SSsSsSSSsSSLLL

LTL

KS

(Q5A

AP9

)3

311

389536

389370

3600

0plusmn

4100

2600

0plusmn

3100

00034

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

314

393914

394074

3600

0plusmn

2400

01700

0plusmn

1800

000026

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

346

411551

411679

3100

0plusmn

2000

01700

0plusmn

7200

0017

KTV

CNWQtLGHTD

EFEE

stQFA

RGVS

DtAL

VGGITKL

(Q59K0

1)

3

218

411482

TCG

KSCF

INSttANKL

IsYN

LFQSStEVDS

IGPT

GSK

T(Q

59KZ

4)386

41162

KTT

PVMGNSsTP

STVTA

NTN

TGAY

STSsDt

AAKP

TKKA

TKR

(A0A

0A6JKT

0)391

411504

RILLL

LPLL

GVtILTS

KSSLES

ISLN

sPSD

SIALssSKS

(Q5A

H83)

234

441651

441518

2900

0plusmn

2700

01600

0plusmn

1100

010

KST

SSANIKtKPK

PNTA

TtAT

TPTA

tTAT

TtA

TTSSID

PTEK

D(A

0A0A

4CH02)

3259

442103

441999

3200

0plusmn

1900

01700

0plusmn

7200

0080

RLS

GtN

NPG

sGsG

sGGGGGGGANNNSLPG

YSV

GSSIG

RGRG

LGRG

(A0A

0A6LWB8

)3

189

436859

436693

3300

0plusmn

2100

07100plusmn

1300

0020

KNYN

QFK

LIEL

DDSM

NTT

TTTT

TTTT

tTttT

tTST

IGKF

(A0A

0A6M

D82)

3376

436787

KSK

sTTIHNQSN

IHSE

QISIN

DEN

NNKS

tstS

TSTD

TKK

(A0A

0A3E

QM3)

132

437257

437082

9600plusmn

4600

5400plusmn1800

0058

KSSGGSSDTK

SVWIAtTGSD

FASQ

SNSD

SsStASR

NSSSSASR

Q(A

0A0A

3BP13)

3246

437317

KEsEG

VWGWsLQCL

LLLF

TKVNLR

scDQL

LICA

LVIRE

(Q5A

KS1)

299

465996

466136

1500

0plusmn

1100

3200plusmn

2700

000

014

KVILIG

NSM

TsRT

TsSV

RFFSIM

LTVS

LPItN

ILVsSEL

SKV

(A0A

0A3B

Z01)

3311


Table 2 Proteins identified from a miniaturized assay of HTN5 treatment of C albicans with increased MS signals Average MS signalsand their standard deviations were calculated based on six replicates Proteins indicated with an asterisk (lowast) have been determined usingtheir associated protein families Lower case letters in amino acid sequences indicated modified residues Blank spaces indicated proteinsunidentified by the databases

Biologicalfunction

Observedprecursor119898119911

Theoreticalprecursor119898119911

Ion counts119901 values Amino acid sequence Protein name Charges 119883CorrBefore After

Surface-associated 129759 129854 18000 plusmn

880051000 plusmn5400 000042 KKPsTEDtFSKY

Peroxisomal proteinfamilylowast

(A0A0A6JC81)1 182

Mitochondrial-associated 129759 129749 18000 plusmn

880051000 plusmn5400 000042 RNsIttEsVKA

Aldehyde hydrogenasefamilylowast

(A0A0A3BNV6)1 203

DNA binding

57544 57645 9700 plusmn1000

28000 plusmn6200 000055 KSLLSRI

Telomerase reversetranscriptase familylowast

(C4YDL3)1 154

129759

12977518000 plusmn8800

51000 plusmn5400 000042

KNVIPAtItKV Spt6(D3IZV2)

1

154

129867 KEFTIGPFKcIKW

Transcriptionalregulatory protein

familylowast(A0A0A3CXJ5)

156

Miscellaneous57544 57631 9700 plusmn

110028000 plusmn6200 000055 RSLNSRI ATPase familylowast

(A0A0A4CD62) 1 315

190171 190374 7800 plusmn3900

13000 plusmn2300 0025 KNKATssSSStsRDTRW Leucine rich repeatslowast

(C4YMX3) 2 319

Uncharacterized 129759 129769 18000 plusmn8800

51000 plusmn5400 000042 RNNTtVSKGRIKV (A0A0A6M207) 1 151

129770 KKHQtHILNVKS (A0A0A6HZ28) 160

In another previous study quantitative LC MS wasperformed to characterize changes in the mitochondrialproteome of C albicans upon HTN5 treatments [33] Theauthors reported an upregulation of mitochondrial proteinsmainly involved in genomemaintenance and gene expressionand a downregulation for respiratory enzyme complexesIn our work the MS analysis was performed on samplesresulted from the tryptic shaving of adhered intact cellsGiven that the two approaches focused on different parts ofthe proteome we should not directly compare the proteinsidentified from these studies It suffices to conclude that ageneral decrease in MS signals was observed in our workfor many virulence- and surface-associated proteins afterHTN5 treatment as onewould expect Finally readers shouldbe reminded that the peptide sample quantity producedfrom our miniaturized technique was substantially lowerthan that typically used in previous studies due to thelower amount of starting materials in our work Neverthe-less one could alleviate this limitation by increasing thedensity of adhered cells on glass with a cell suspension ofhigher concentration andor by increasing the surface areaused of each assay which is defined by the hydrophobicboundary made by the user An increase in cell numberper assay will directly increase the quantity of peptidesproduced for LC MS analysis and subsequently improve thenumber of proteins identified andor the confidence of theidentification

4 Conclusions

This paper reported successful miniaturization of the trypticdigestion of surface proteins performed on whole cellsIn particular C albicans were adhered on glass surfacewhere introduction and removal of reagent solutions wereconveniently conducted with a micropipette Compared tothe use of cells suspended in solutions our treatment ofadhered cells also better resembles the antifungal treat-ment of C albicans cells adhered on tissues in the oralcavity Importantly the reduction in reagent volumes frommL to 120583L levels will allow users to perform many morereplicate assays while keeping an overall low consump-tion of reagents and cells Nevertheless the downside ofthe miniaturization is the reduced peptide sample quantityresulting from the LC MS analysis which could reducethe confidence of the protein identification Ultimatelyresearchers should select methods based on their purposesIn this case the miniaturized approach with adhered cellsis more effective for high-throughput screenings of celltypes or fungicides while the batch mode with suspendedcells is more suitable for a comprehensive profiling of theproteome

Disclosure

The paper is a part of MS thesis work of S Fan [34] WalterL Siqueira andKenK-C Yeung areCoprincipal Investigators


Competing Interests

The authors declare that there is no conflict of interests

Acknowledgments

The work was funded by the University of Western Ontariothe Natural Sciences and Engineering Research Council ofCanada (NSERC) and the Canadian Institutes of HealthResearch (CIHR) Walter L Siqueira is recipient of CIHRNew Investigator Salary Award The authors acknowledgeMs Kristina Jurcic for assistance in MALDI MS data acqui-sition and processing and Ms Tayebeh Basiri for insightfuladvices in fungal culturing Permission to access the Univer-sity of Western Ontario MALDI Mass Spectrometry Facilityis also acknowledged

References

[1] P Sudbery N Gow and J Berman ldquoThe distinct morphogenicstates of Candida albicansrdquo Trends in Microbiology vol 12 no7 pp 317ndash324 2004

[2] D W Williams T Kuriyama S Silva S Malic and M A OLewis ldquoCandida biofilms and oral candidosis treatment andpreventionrdquo Periodontology 2000 vol 55 no 1 pp 250ndash2652011

[3] C J Seneviratne L Jin and L P Samaranayake ldquoBiofilmlifestyle of Candida a mini reviewrdquo Oral Diseases vol 14 no7 pp 582ndash590 2008

[4] A Sanguineti J K Carmichael andK Campbell ldquoFluconazole-resistant Candida albicans after long-term suppressive therapyrdquoArchives of Internal Medicine vol 153 no 9 pp 1122ndash1124 1993

[5] D Vukosavljevic W Custodio A A Del Bel Cury and W LSiqueira ldquoThe effect of histatin 5 adsorbed on PMMA andhydroxyapatite on Candida albicans colonizationrdquo Yeast vol29 no 11 pp 459ndash466 2012

[6] W L Siqueira W Zhang E J Helmerhorst S P Gygi and F GOppenheim ldquoIdentification of protein components in in vivohuman acquired enamel pellicle using LC-ESI-MSMSrdquo Journalof Proteome Research vol 6 no 6 pp 2152ndash2160 2007

[7] D Vukosavljevic W Custodio and W L Siqueira ldquoSalivaryproteins as predictors and controls for oral healthrdquo Journal ofCell Communication and Signaling vol 5 no 4 pp 271ndash2752011

[8] J M Laudenbach and J B Epstein ldquoTreatment strategies fororopharyngeal candidiasisrdquo Expert Opinion on Pharmacother-apy vol 10 no 9 pp 1413ndash1421 2009

[9] F G Oppenheim Y C Yang R D Diamond D Hyslop G DOffner and R F Troxler ldquoThe primary structure and functionalcharacterization of the neutral histidine-rich polypeptide fromhuman parotid secretionrdquo The Journal of Biological Chemistryvol 261 no 3 pp 1177ndash1182 1986

[10] J O TenovuoHuman Saliva Clinical Chemistry andMicrobiol-ogy CRC Press 1989

[11] F G Oppenheim T Xu F M McMillian et al ldquoHistatinsa novel family of histidine-rich proteins in human parotidsecretion Isolation characterization primary structure andfungistatic effects onCandida albicansrdquoThe Journal of BiologicalChemistry vol 263 no 16 pp 7472ndash7477 1988

[12] E J Helmerhorst P Breeuwer W Van rsquot Hof et al ldquoThecellular target of histatin 5 on Candida albicans is the energized

mitochondrionrdquo The Journal of Biological Chemistry vol 274no 11 pp 7286ndash7291 1999

[13] A Freiwald and S Sauer ldquoPhylogenetic classification and iden-tification of bacteria by mass spectrometryrdquo Nature protocolsvol 4 no 5 pp 732ndash742 2009

[14] G Marklein M Josten U Klanke et al ldquoMatrix-assisted laserdesorption ionization-time of flight mass spectrometry for fastand reliable identification of clinical yeast isolatesrdquo Journal ofClinical Microbiology vol 47 no 9 pp 2912ndash2917 2009

[15] S Sauer and M Kliem ldquoMass spectrometry tools for theclassification and identification of bacteriardquo Nature ReviewsMicrobiology vol 8 no 1 pp 74ndash82 2010

[16] C Liu S A Hofstadler J A Bresson et al ldquoOn-line dualmicro-dialysis with ESI-MS for direct analysis of complex biologicalsamples and microorganism lysatesrdquo Analytical Chemistry vol70 no 9 pp 1797ndash1801 1998

[17] T C Cain D M Lubman and W J Weber ldquoDifferentiationof bacteria using protein profiles from matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 8 no 12 pp 1026ndash1030 1994

[18] Y-P Ho and P M Reddy ldquoIdentification of pathogens by massspectrometryrdquo Clinical Chemistry vol 56 no 4 pp 525ndash5362010

[19] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013

[20] J P Anhalt and C Fenselau ldquoIdentification of bacteria usingmass spectrometryrdquoAnalytical Chemistry vol 47 no 2 pp 219ndash225 1975

[21] S Vaidyanathan J J Rowland D B Kell and R GoodacreldquoDiscrimination of aerobic endospore-forming bacteria viaelectrospray-ionization mass spectrometry of whole cell sus-pensionsrdquo Analytical Chemistry vol 73 no 17 pp 4134ndash41442001

[22] M Welker ldquoProteomics for routine identification of microor-ganismsrdquo Proteomics vol 11 no 15 pp 3143ndash3153 2011

[23] R DHolland J GWilkes F Rafii et al ldquoRapid identification ofintact whole bacteria based on spectral patterns using matrix-assisted laser desorptionionization with time-of- flight massspectrometryrdquo Rapid Communications in Mass Spectrometryvol 10 no 10 pp 1227ndash1232 1996

[24] E B Moffa M C M Mussi Y Xiao et al ldquoHistatin 5inhibits adhesion of C albicans to reconstructed human oralepitheliumrdquo Frontiers inMicrobiology vol 6 article 885 pp 1ndash72015

[25] M J Rodrıguez-Ortega N Norais G Bensi et al ldquoCharacteri-zation and identification of vaccine candidate proteins throughanalysis of the group A Streptococcus surface proteomerdquoNatureBiotechnology vol 24 no 2 pp 191ndash197 2006

[26] F Doro S Liberatori M J Rodrıguez-Ortega et al ldquoSurfomeanalysis as a fast track to vaccine discovery identification of anovel protective antigen for group B Streptococcus hyperviru-lent strain COH1rdquoMolecular and Cellular Proteomics vol 8 no7 pp 1728ndash1737 2009

[27] A Gil-Bona C M Parra-Giraldo M L Hernaez et al ldquoCan-dida albicans cell shaving uncovers new proteins involved incell wall integrity yeast to hypha transition stress response andhost-pathogen interactionrdquo Journal of Proteomics vol 127 pp340ndash351 2015


[28] M L Hernaez P Ximenez-Embun M Martınez-Gomariz MD Gutierrez-Blazquez C Nombela and C Gil ldquoIdentificationof Candida albicans exposed surface proteins in vivo by a rapidproteomic approachrdquo Journal of Proteomics vol 73 no 7 pp1404ndash1409 2010

[29] V Vialas P Perumal D Gutierrez et al ldquoCell surface shavingof Candida albicans biofilms hyphae and yeast form cellsrdquoProteomics vol 12 no 14 pp 2331ndash2339 2012

[30] M R Insenser M L Hernaez C Nombela M Molina GMolero and C Gil ldquoGel and gel-free proteomics to identifySaccharomyces cerevisiae cell surface proteinsrdquo Journal of Pro-teomics vol 73 no 6 pp 1183ndash1195 2010

[31] C Gyurko U Lendenmann E J Helmerhorst R F Troxlerand F GOppenheim ldquoKilling of Candida albicans by histatin 5cellular uptake and energy requirementrdquoAntonie van Leeuwen-hoek vol 79 no 3-4 pp 297ndash309 2001

[32] A Pitarch M Sanchez C Nombela and C Gil ldquoSequentialfractionation and two-dimensional gel analysis unravels thecomplexity of the dimorphic fungus Candida albicans cell wallproteomerdquo Molecular amp Cellular Proteomics vol 1 no 12 pp967ndash982 2002

[33] T Komatsu E Salih E J Helmerhorst G D Offner and FG Oppenheim ldquoInfluence of histatin 5 on Candida albicansmitochondrial protein expression assessed by quantitative massspectrometryrdquo Journal of Proteome Research vol 10 no 2 pp646ndash655 2011

[34] httpirlibuwocaetd3243

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


complexity of the proteome and background of lysed cellssample cleanup andor fractionation is crucial to obtaininghigh qualityMSdata [16]The identification of a large numberof proteins also requires a substantial effort in data analysisAlternatively the analysis of intact whole cells offers thebenefit of reduced complexity Anhalt and Fenselau wereamong the earliest to report the analysis of whole cellsfor species identification [20] Through gentle heating inthe ion source phospholipids were vaporized leading tothe production of characteristic mass spectra Alternativelyspecies identification through proteins and peptides fromwhole cell analyses has also been reported [21ndash23]

When applying MS to study changes in protein levels ofC albicans upon treatment with HTN5 the global approachof studying the total proteome was certainly appropriateHowever our group previously observed a reduction incell adhesion property upon administration of HTN5 in90 minutes suggesting an effect of HTN5 on the surfaceproteins [24] Hence the targeted approach on the surfaceproteins is logical for studying the influence of HTN5 In2006 Rodrıguez-Ortega et al reported a protocol calledldquocell shavingrdquo which allowed them to focus on proteinspartially exposed on the outside of cells [25] This was doneby subjecting whole live cells to proteolytic enzymes in asolution The surface peptides cleaved from intact cells weresubsequently isolated for MS analyses Others have alsoused a similar approach towards studying surface proteinsfrom other fungal and bacterial species [26 27] Despite itseffectiveness the procedure of ldquocell shavingrdquo is still generallytime-consuming Cells grown in media were harvested andwashed followed by overnight tryptic digestion for 18ndash20hours in mL volume solution The sample mixture was thencentrifuged and the supernatant was collected for fraction-ation andor enrichment prior to injection into the liquidchromatography-mass spectrometer [28ndash30] Furthermoreto overcome biological variation and growth variability theentire procedure was typically repeated for multiple timesthus requiring large quantities of cells and reagents

In this study the aim is to refine and miniaturize theexisting cell shaving technique by performing the procedureson adhered C albicans cells rather than suspended cells insolution We will demonstrate that the attachment of cells onsurfaces allows easy introduction and removal of 120583L volumereagents solutions with a micropipette Furthermore treat-ment with antifungal peptide HTN5 is readily performedon the adhered cells We will also demonstrate that the 120583Lsample volume resulting from our miniaturized approachis directly compatible with subsequent MALDI MS andorLC ESI MSMS analyses Importantly the resulting MS datashould be comparable to previous information obtained withthe conventional in solution approach that required muchlarger quantities of reagent and cells

2 Materials and Methods

21 C albicans Culturing The strain of C albicans used forall experiments was ATCC90028 Stock cultures of cells weregrown on plates containing Sabouraud dextrose agar (SDA)medium (Becton Dickson and Company NJ USA) The

(a)

(b)

(c)

Figure 1 Schematics of the miniaturized procedures of cell adhe-sion treatment and tryptic digestion Description is presented inMaterials and Methods

inoculated plates were incubated at 37∘C for 48 hours A thirdof a loop of the colonies were transferred into 45mL of yeastnitrogen base (YNB) supplemented with 50mM sucrose(Sigma Aldrich MO USA) The mixture was then incubatedat 37∘C for 21 hours under shaking at 200 rpm Following theincubation the mixture was centrifuged at 6000 rpm for 5minutes The pellet was resuspended in phosphate bufferedsaline solution (PBS pH 74 Sigma Aldrich) The final cellsuspension was stored at 4∘C until use

22 Adhesion of C albicans to Glass Slides and TreatmentwithHTN5 ADakodelimiting pen composed of 60ndash100 1-bromopropane and 5ndash10 dipentene (Cedarlane BurlingtonCA) was used to draw circles with an inner diameter ofroughly 6mm on standard microscope glass slides (Fig-ure 1(a)) The hydrophobic ink functioned as a barrier tosurround a droplet of aqueous sample solution A total ofthree circles were made on each glass slide (Figure 1(b))During an experiment each sample spot was covered with aninverted glass vial containing a wet cotton which maintaineda high humidity environment and minimized evaporation ofthe sample droplet (Figure 1(c)) To prepare for cell adhesionthe glass surface within the hydrophobic circles was treatedwith 25 120583L of 010mgmL human serum albumin (HSA)solution (Sigma Aldrich) for 2 hours at room temperature(RT) based on a protocol previously reported by our group[5] The HSA-coated surface was then washed three timeswith water Next 25120583L of C albicans suspension at aconcentration of 107 cellsmL was introduced to the HSA-coated slideThe adhesion was carried out under a 90-minute













2 120583m

(a) Control

2 120583m

(b) 30120583gmL

2 120583m

(c) 25120583gmL

2 120583m

(d) 20120583gmL
















ble1Proteins

identifi

edfro

mam

iniaturiz

edassayof

HTN

5tre

atmento

fCalbica

nswith

decreasedMSsig

nals

AverageM

Ssig

nalsandtheirstand

arddeviations

werec

alculatedbased

onsix


icated

with

anasteris

k(lowast)h

aveb

eendeterm

ined


proteinfamiliesLow

ercaselette

rsin

aminoacidsequ

encesind

icated

mod

ified

resid

ues

Blankspaces

indicatedproteins

unidentifi

edby

thed

atabases

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Virulence

128483

128665

2700

0plusmn

9500

1400

0plusmn

2800

0016

KsLYT

ISPN

KGKR

Transcrip

tionalregulator

STP3

(A0A

0A6LWB8

)3

252

128369

KSK

YNLP

FAMKE

Cand

idapepsin

-7(A

0A0A

4B714)

375

129587

129570

6600

0plusmn

1100

1900

0plusmn

7000

000

0013

RtKKW

GLG

WIK

NIntegrasefam

ilylowast

(A0A

0A3B

ML7

)1

161

357979

358147

4000

0plusmn

5000

3100

0plusmn

3600

0030

HPQ

YsEA

CsAV

mVVTY

SSGSG

EHIH

TTDIK

Kexin

(A0A

0A6IYX

0)3

333

389536

389280

3600

0plusmn

4100

2600

0plusmn

3100

00037

ELKT

TVIV

TSCF

NNVC

SETsITTPK

tAVtAT

tSK

Flocculinlowast

(A0A

0A3C

M71)

3310

465996

465822

1500

0plusmn

1100

3200plusmn

2700

000

014

KHLT

LKSSTP

AST

LEYS

TSIPPA

LATT

SSSLStES

TtLttIS

RS

325

411551

411594

5100

0plusmn

1200

02400

0plusmn

1700

00017

VTF

VEK

AtST

STTN

tTttT

TTTT

TTTT

TTT

TIPV


ilylowast

(A0A

0A6K

6B3)

3323

412103

412205

3100

0plusmn

2300

01700

0plusmn

7200

013

KNVKV

ITTT

TTtSPS

SFSSSSSLMsPITPQ

TPN

IPKT

PKT

PXdo

mainlowast

(A0A

0A3C

BE8)

3394

Surfa

ce-

associated

117588

117366

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

RtIN

LDsQ

VKY

31015840(21015840 )51015840-Bisp

hosphate

nucle

otidase2

(A0A

0A6N

TV1)

1316

465996

465987

6600

0plusmn

1100

2000

0plusmn

7000

000

001

mQTS

ISttT

IEDHLH

HYsPE

ESQKL

LSRE

SSIN

TDLF

KE

Budsites

electionprotein

BUD4

(A0A

0A3D

IG6)

3313

Mito

chon

drial-

associated

117588

117515

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KYM

LLTL

LtKL

YAP-bd

ALF

4glom

ulin

familylowast

(A0A

0A3C

AC1)

1296

129587

129768

6600

0plusmn

1100

1900

0plusmn

7000

000

001

KLT

tLISSIEN

KI

296

137401

137670

1600

0plusmn

1300

1000

0plusmn

3000

000

47RTA

SGNIIPS

STGAAKA

Glyceraldehyde-3-ph

osph

ate

dehydrogenase

(Q92211)

1286

465996

466023

1500

0plusmn

1100

3200plusmn

2700

000

014

KStIV

EEIYsN

ARS

HLV

QGNKE

mGmALF

NE

LLAIN

ESIYGKV

Clusteredmito

chon

dria

proteinho

molog

(A0A

0A4C

6G1)

3337

DNAbind

ing

80477

80444

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQPA

VFSRI

DNApo

lymerase

(Q5A

4E5)

1256

128483

128353

2700

0plusmn

9500

1400

0plusmn

2800

0016

RVA

SHsLsTsR

RMinichrom

osom

emaintenance

protein10

(A0A

0A3C

IN8)

1267

333128

333142

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

KGFT

NTM

ISHIG

FDPT

GtsL

NQNsTS

LGsK

S3

321

280676

280620

4700

0plusmn

3600

03000

0plusmn

2300

0021

RTT

PPTV

sITG

PNPsSSPA

sAST

NtSKS

Phosph

atasefam

ilylowast

(A0A

0A3B

QA3)

3360


ble1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

337660

337698

4300

0plusmn

1200

03200

0plusmn

2800

0023

KSSPT

SsAT

TTAT

tSVsISSLSLtMGKP

KNSK

LMediatorc

omplexlowast

(A0A

0A3C

P70)

3315

349815

350080

3400

0plusmn

8900

1800

0plusmn

1600

00054

KLG

SVLT

tRSQ

LIEY

ELtTRtIFIN

CSA

ALK

ISpt6

(D3IZV

2)3

311

350340

350474

4300

0plusmn

8300

3100

0plusmn

2500

0014

REV

IIKL

SITR

tHtPPP

DST

tTTT

PTTS

IEKT

Znfin

gerd

omainlowast

(A0A

0A6J6A

5)3

357

393914

394046

3600

0plusmn

4000

1700

0plusmn

8000

00026

VSSY

ILDGNNST

KLPsPV

LtHtTF

DSR

sDEG

QR

Basic

leucinez

ipperd

omainlowast

(A0A

0A3D

LJ5)

3399

411551

411695

5100

0plusmn

1200

02400

0plusmn

1700

00017

RAPQ

sIQLP

PIQsFtKsQ

AVFP

QSV

RDSA

PAANFN

RY

Transcrip

tionfactor

andDNA

bind


(A0A

0A6ITE

8)3

312

465996

465915

1500

0plusmn

1100

3200plusmn

2700

000

014

MKImmIPTH

HQtYNIN

THQPP

QQHQYL

PPPG

tSY

TSPR

A312

411551

411487

5100

0plusmn

1200

02400

0plusmn

1700

00017

KsStQ

MSSCtNsV

TQTL

DRL

PKIV

STQQNNL

TPTS

KI

Zn(2)-DNAbind

ingdo

mainlowast

(A0A

0A6K

Y81)

3303

465996

466019

1500

0plusmn

1100

3200plusmn

2700

000

014

RRS

VSYS

PGPsSIKS

QLP

HLT

SSST

TtssVQ

SPP

PPPP

SQPP

RG

361

417360

417373

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

KtV

TsIN

GSP

PPLE

tAPsSH

HNVPIDFIHFK

KESD

RT

Uncharacterized

protein

(A0A

0A3E

PD4)

3312

436859

436789

3300

0plusmn

2100

07100plusmn

1300

0020

KNMQFP

PYQVS

sHNsSEtSQ

sIPN

TPSITR

QVE

SNTR

S

Transcrip

tionalregulatory

proteinLE

U3

(A0A

0A3B

TY8)

3354

436790

KtPTT

TTTT

TTtANGNTS

NGNTS

NGNsTGKT

ATA

ATAT

KSNtK

Transcrip


(A0A

0A3C

3Y1)

319

Protein

synthesis

80477

80541

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQTS

LNDKC

Manno

syltransfe

rase

familylowast

(A0A

0A3C

UB4

)1

254

80445

RVKI

DSD

KS

ProteintransporterS

EC24

(A0A

0A3E

HG4)

151

117588

117398

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KTK

QFN

DsK

KK

Vesic

letetheringprotein

familylowast

(A0A

0A6K

IG6)

1201

128483

128372

2700

0plusmn

9500

1400

0plusmn

2800

0016

KtAGsN

HNsEKK

Ribo

somalRN

A-processin

gprotein12

(A0A

0A4B

GJ7)

1251

412103

411993

3100

0plusmn

2300

01700

0plusmn

7200

013

KFF

sDNIANDLA

tTTT

TTTT

TNTG

AtSV

HPIL

QVDAIK

YCA

SCS


(A0A

0A6L

8P2)

3324

436859

437093

3300

0plusmn

2100

07100plusmn

1300

0020

KES

SSTA

DQPS

VVPP

QES

HKD

TVET

PKPE

VtEtsV

EAtKE

Transla


4G(A

0A0A

4CDK4

)3

299

436703

KNPT

PTPT

PTPT

PTPN

NLA

QGVDsSST

LDV

EtTL

tGLtRR

I


sphate

synthase

cycla

sesubu

nit

(A0A

0A6I238)

321


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Sign

allin

g

350340

350460

4300

0plusmn

8300

3100

0plusmn

2500

0013

KRS

SITtPtPP

LTTT

HSSNGNGNGNV

NVNVNsK

R

Peroxisometransmem

brane

receptorlowast

(A0A

0A3Z

WC2

)3

330

411551

411676

3100

0plusmn

2000

01700

0plusmn

7200

0017

KQST

TNTsTL

ssTtAAST

LATS

NNTQ

PDTY

TSTS

TSIRG

Pleckstrin

homologydo

mainlowast

(A0A

0A6L

5L9)

3333

441651

441487

2900

0plusmn

2700

01600

0plusmn

1100

010

RQHPD

PLSN

QsN

FNsN

TINNYS

NYR

SsTRS

GLD

PsQRH

RhoGTP

asea

ctivating

proteindo

mainlowast

(A0A

0A3B

MU5)

3412

465996

466032

1500

0plusmn

1100

3200plusmn

2700

000

014

KDLP

IGYILH

mIN

LcPN

IVsLNLG

NLSLsTD

YEISRS

TIHKY

F-bo

xproteinCO

S111

(A0A

0A3C

YY6)

3368

465821

KWNKE

KIEL

DsPLIVS

YVSSLC

NGGGGGIIT

NsTNST

ttNSK

Pentatric

opeptid

erepeat

(PPR

)protein

familylowast

(A0A

0A3C

GE7

)111

Misc

ellaneou

s

128483

128542

2700

0plusmn

9500

1400

0plusmn

2800

0016

RNDsD

tsLsK

EInsulin

indu

cedprotein

familylowast

(A0A

0A3C

RM0)

116

6RYtmNEV

FKV

GYF

domainlowast

(A0A

0A3C

0N8)

333128

333159

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

ETsQ

FMGNAES

EDLtGNGLL

TSTL

AVLS

SIS

Orfin

Calbicans

major

repeat

sequ

enceR

B2region

(Q5A

475)

3315

333128

333244

6300

0plusmn

1200

1400

0plusmn

1400

023

KLS

SLGNHGTtTT

SSLS

SSsSsSIsNNT

SIAKI

CBSdo

mainlowast

(A0A

0A6JTB

4)3

346

337660

337659

4300

0plusmn

1200

03300

0plusmn

2800

0RKQ

QDQNEV

AGAAAAT

TTTA

tAtAT

AAT

NWKP

KN

321

349815

349857

3400

0plusmn

8900

1800

0plusmn

1600

00054

KSSsLIK

NsTsSNQSsPA

TSTN

TSIV

DVPIEK


(A0A

0A3C

A48)

3321

417360

417295

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

RIN

NNNDKs

sILsNITTT

NTT

TGTG

TTNTtT

VPS

IKTK

R(A

0A0A

6IY5

3)424

349815

349948

3400

0plusmn

8900

1800

0plusmn

1600

00054

KAFS

SsKL

TSDSA

NStNStNsTSM

SILG

NDKD

CDKinhibitorP

HO81

(A0A

0A6M

Y31)

3316

389536

389486

3600

0plusmn

4100

2600

0plusmn

3100

00037

RISRP

NGVG

GISTS

GSSSP

TTEF

VTP

QAs

KsSV

DQNKK

RUbiqu


(C4Y

NW3)

3329

442103

442109

3200

0plusmn

1900

01700

0plusmn

7200

0080

RHLILG

YKItV

VtDHQSLTsVMTS

SSRP

ENNRM

IRW

Aspartic

peptidasefam

ilylowast

(A0A

0A4C

VF8

)3

383

393914

394016

3600

0plusmn

2400

01700

0plusmn

1800

000026

RNVNGSG

StNtN

TMtRLD

sTTIASSLF

CRQLY

FNLL

SKD

Globinfamilylowast

(A0A

0A4C

D75)

3311

437257

437390

9600plusmn

4600

5400plusmn

1800

0058

RNNsT

VSST

NSL

mSN

NsD

TNtAAT

AAT

AAT

SGS

TTNNVKR

M(A

0A0A

3XE4

6)338


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

441615

441482

2900

0plusmn

2700

01600

0plusmn

1100

010

RNGtYSSsStSSSsTSSVS

SSSA

TTANGES

LNST

THNIQ

LERT

Sec2plowast

(A0A

0A6JGN5)

3289

465996

466133

1500

0plusmn

1100

3200plusmn

2700

000

014

KQNYV

DPG

QIAIKGLK

GFV

NLG

AtCF

MssILQtLIH

NPL

IKY

Ubiqu

itincarboxyl-te

rminal

hydrolase

(C4Y

KS7)

3369

466093

KYD

KPmED

TEEIDDVtSISK

sINEQ

IDDPF

sQFN

SVTL

RY

Protein-serin

ethreon

ine

kinase

(A0A

0A4A

NB2

)35

1

Uncharacterized

117588

117373

6400

0plusmn

4000

2200

0plusmn

1100lt000

001

KtIm

tItIK

I(Q

59T3

9)1

207

333128

333260

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

RIsNIITL

NSSLS

SSsSsSSSsSSLLL

LTL

KS

(Q5A

AP9

)3

311

389536

389370

3600

0plusmn

4100

2600

0plusmn

3100

00034

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

314

393914

394074

3600

0plusmn

2400

01700

0plusmn

1800

000026

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

346

411551

411679

3100

0plusmn

2000

01700

0plusmn

7200

0017

KTV

CNWQtLGHTD

EFEE

stQFA

RGVS

DtAL

VGGITKL

(Q59K0

1)

3

218

411482

TCG

KSCF

INSttANKL

IsYN

LFQSStEVDS

IGPT

GSK

T(Q

59KZ

4)386

41162

KTT

PVMGNSsTP

STVTA

NTN

TGAY

STSsDt

AAKP

TKKA

TKR

(A0A

0A6JKT

0)391

411504

RILLL

LPLL

GVtILTS

KSSLES

ISLN

sPSD

SIALssSKS

(Q5A

H83)

234

441651

441518

2900

0plusmn

2700

01600

0plusmn

1100

010

KST

SSANIKtKPK

PNTA

TtAT

TPTA

tTAT

TtA

TTSSID

PTEK

D(A

0A0A

4CH02)

3259

442103

441999

3200

0plusmn

1900

01700

0plusmn

7200

0080

RLS

GtN

NPG

sGsG

sGGGGGGGANNNSLPG

YSV

GSSIG

RGRG

LGRG

(A0A

0A6LWB8

)3

189

436859

436693

3300

0plusmn

2100

07100plusmn

1300

0020

KNYN

QFK

LIEL

DDSM

NTT

TTTT

TTTT

tTttT

tTST

IGKF

(A0A

0A6M

D82)

3376

436787

KSK

sTTIHNQSN

IHSE

QISIN

DEN

NNKS

tstS

TSTD

TKK

(A0A

0A3E

QM3)

132

437257

437082

9600plusmn

4600

5400plusmn1800

0058

KSSGGSSDTK

SVWIAtTGSD

FASQ

SNSD

SsStASR

NSSSSASR

Q(A

0A0A

3BP13)

3246

437317

KEsEG

VWGWsLQCL

LLLF

TKVNLR

scDQL

LICA

LVIRE

(Q5A

KS1)

299

465996

466136

1500

0plusmn

1100

3200plusmn

2700

000

014

KVILIG

NSM

TsRT

TsSV

RFFSIM

LTVS

LPItN

ILVsSEL

SKV

(A0A

0A3B

Z01)

3311



Biologicalfunction







(A0A0A6JC81)1 182




(A0A0A3BNV6)1 203

DNA binding

57544 57645 9700 plusmn1000



(C4YDL3)1 154

129759

12977518000 plusmn8800

51000 plusmn5400 000042


1

154




156



(A0A0A4CD62) 1 315

190171 190374 7800 plusmn3900


(C4YMX3) 2 319





4 Conclusions


Disclosure



Competing Interests


Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













2 120583m

(a) Control

2 120583m

(b) 30120583gmL

2 120583m

(c) 25120583gmL

2 120583m

(d) 20120583gmL
















ble1Proteins

identifi

edfro

mam

iniaturiz

edassayof

HTN

5tre

atmento

fCalbica

nswith

decreasedMSsig

nals

AverageM

Ssig

nalsandtheirstand

arddeviations

werec

alculatedbased

onsix


icated

with

anasteris

k(lowast)h

aveb

eendeterm

ined


proteinfamiliesLow

ercaselette

rsin

aminoacidsequ

encesind

icated

mod

ified

resid

ues

Blankspaces

indicatedproteins

unidentifi

edby

thed

atabases

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Virulence

128483

128665

2700

0plusmn

9500

1400

0plusmn

2800

0016

KsLYT

ISPN

KGKR

Transcrip

tionalregulator

STP3

(A0A

0A6LWB8

)3

252

128369

KSK

YNLP

FAMKE

Cand

idapepsin

-7(A

0A0A

4B714)

375

129587

129570

6600

0plusmn

1100

1900

0plusmn

7000

000

0013

RtKKW

GLG

WIK

NIntegrasefam

ilylowast

(A0A

0A3B

ML7

)1

161

357979

358147

4000

0plusmn

5000

3100

0plusmn

3600

0030

HPQ

YsEA

CsAV

mVVTY

SSGSG

EHIH

TTDIK

Kexin

(A0A

0A6IYX

0)3

333

389536

389280

3600

0plusmn

4100

2600

0plusmn

3100

00037

ELKT

TVIV

TSCF

NNVC

SETsITTPK

tAVtAT

tSK

Flocculinlowast

(A0A

0A3C

M71)

3310

465996

465822

1500

0plusmn

1100

3200plusmn

2700

000

014

KHLT

LKSSTP

AST

LEYS

TSIPPA

LATT

SSSLStES

TtLttIS

RS

325

411551

411594

5100

0plusmn

1200

02400

0plusmn

1700

00017

VTF

VEK

AtST

STTN

tTttT

TTTT

TTTT

TTT

TIPV


ilylowast

(A0A

0A6K

6B3)

3323

412103

412205

3100

0plusmn

2300

01700

0plusmn

7200

013

KNVKV

ITTT

TTtSPS

SFSSSSSLMsPITPQ

TPN

IPKT

PKT

PXdo

mainlowast

(A0A

0A3C

BE8)

3394

Surfa

ce-

associated

117588

117366

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

RtIN

LDsQ

VKY

31015840(21015840 )51015840-Bisp

hosphate

nucle

otidase2

(A0A

0A6N

TV1)

1316

465996

465987

6600

0plusmn

1100

2000

0plusmn

7000

000

001

mQTS

ISttT

IEDHLH

HYsPE

ESQKL

LSRE

SSIN

TDLF

KE

Budsites

electionprotein

BUD4

(A0A

0A3D

IG6)

3313

Mito

chon

drial-

associated

117588

117515

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KYM

LLTL

LtKL

YAP-bd

ALF

4glom

ulin

familylowast

(A0A

0A3C

AC1)

1296

129587

129768

6600

0plusmn

1100

1900

0plusmn

7000

000

001

KLT

tLISSIEN

KI

296

137401

137670

1600

0plusmn

1300

1000

0plusmn

3000

000

47RTA

SGNIIPS

STGAAKA

Glyceraldehyde-3-ph

osph

ate

dehydrogenase

(Q92211)

1286

465996

466023

1500

0plusmn

1100

3200plusmn

2700

000

014

KStIV

EEIYsN

ARS

HLV

QGNKE

mGmALF

NE

LLAIN

ESIYGKV

Clusteredmito

chon

dria

proteinho

molog

(A0A

0A4C

6G1)

3337

DNAbind

ing

80477

80444

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQPA

VFSRI

DNApo

lymerase

(Q5A

4E5)

1256

128483

128353

2700

0plusmn

9500

1400

0plusmn

2800

0016

RVA

SHsLsTsR

RMinichrom

osom

emaintenance

protein10

(A0A

0A3C

IN8)

1267

333128

333142

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

KGFT

NTM

ISHIG

FDPT

GtsL

NQNsTS

LGsK

S3

321

280676

280620

4700

0plusmn

3600

03000

0plusmn

2300

0021

RTT

PPTV

sITG

PNPsSSPA

sAST

NtSKS

Phosph

atasefam

ilylowast

(A0A

0A3B

QA3)

3360


ble1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

337660

337698

4300

0plusmn

1200

03200

0plusmn

2800

0023

KSSPT

SsAT

TTAT

tSVsISSLSLtMGKP

KNSK

LMediatorc

omplexlowast

(A0A

0A3C

P70)

3315

349815

350080

3400

0plusmn

8900

1800

0plusmn

1600

00054

KLG

SVLT

tRSQ

LIEY

ELtTRtIFIN

CSA

ALK

ISpt6

(D3IZV

2)3

311

350340

350474

4300

0plusmn

8300

3100

0plusmn

2500

0014

REV

IIKL

SITR

tHtPPP

DST

tTTT

PTTS

IEKT

Znfin

gerd

omainlowast

(A0A

0A6J6A

5)3

357

393914

394046

3600

0plusmn

4000

1700

0plusmn

8000

00026

VSSY

ILDGNNST

KLPsPV

LtHtTF

DSR

sDEG

QR

Basic

leucinez

ipperd

omainlowast

(A0A

0A3D

LJ5)

3399

411551

411695

5100

0plusmn

1200

02400

0plusmn

1700

00017

RAPQ

sIQLP

PIQsFtKsQ

AVFP

QSV

RDSA

PAANFN

RY

Transcrip

tionfactor

andDNA

bind


(A0A

0A6ITE

8)3

312

465996

465915

1500

0plusmn

1100

3200plusmn

2700

000

014

MKImmIPTH

HQtYNIN

THQPP

QQHQYL

PPPG

tSY

TSPR

A312

411551

411487

5100

0plusmn

1200

02400

0plusmn

1700

00017

KsStQ

MSSCtNsV

TQTL

DRL

PKIV

STQQNNL

TPTS

KI

Zn(2)-DNAbind

ingdo

mainlowast

(A0A

0A6K

Y81)

3303

465996

466019

1500

0plusmn

1100

3200plusmn

2700

000

014

RRS

VSYS

PGPsSIKS

QLP

HLT

SSST

TtssVQ

SPP

PPPP

SQPP

RG

361

417360

417373

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

KtV

TsIN

GSP

PPLE

tAPsSH

HNVPIDFIHFK

KESD

RT

Uncharacterized

protein

(A0A

0A3E

PD4)

3312

436859

436789

3300

0plusmn

2100

07100plusmn

1300

0020

KNMQFP

PYQVS

sHNsSEtSQ

sIPN

TPSITR

QVE

SNTR

S

Transcrip

tionalregulatory

proteinLE

U3

(A0A

0A3B

TY8)

3354

436790

KtPTT

TTTT

TTtANGNTS

NGNTS

NGNsTGKT

ATA

ATAT

KSNtK

Transcrip


(A0A

0A3C

3Y1)

319

Protein

synthesis

80477

80541

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQTS

LNDKC

Manno

syltransfe

rase

familylowast

(A0A

0A3C

UB4

)1

254

80445

RVKI

DSD

KS

ProteintransporterS

EC24

(A0A

0A3E

HG4)

151

117588

117398

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KTK

QFN

DsK

KK

Vesic

letetheringprotein

familylowast

(A0A

0A6K

IG6)

1201

128483

128372

2700

0plusmn

9500

1400

0plusmn

2800

0016

KtAGsN

HNsEKK

Ribo

somalRN

A-processin

gprotein12

(A0A

0A4B

GJ7)

1251

412103

411993

3100

0plusmn

2300

01700

0plusmn

7200

013

KFF

sDNIANDLA

tTTT

TTTT

TNTG

AtSV

HPIL

QVDAIK

YCA

SCS


(A0A

0A6L

8P2)

3324

436859

437093

3300

0plusmn

2100

07100plusmn

1300

0020

KES

SSTA

DQPS

VVPP

QES

HKD

TVET

PKPE

VtEtsV

EAtKE

Transla


4G(A

0A0A

4CDK4

)3

299

436703

KNPT

PTPT

PTPT

PTPN

NLA

QGVDsSST

LDV

EtTL

tGLtRR

I


sphate

synthase

cycla

sesubu

nit

(A0A

0A6I238)

321


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Sign

allin

g

350340

350460

4300

0plusmn

8300

3100

0plusmn

2500

0013

KRS

SITtPtPP

LTTT

HSSNGNGNGNV

NVNVNsK

R

Peroxisometransmem

brane

receptorlowast

(A0A

0A3Z

WC2

)3

330

411551

411676

3100

0plusmn

2000

01700

0plusmn

7200

0017

KQST

TNTsTL

ssTtAAST

LATS

NNTQ

PDTY

TSTS

TSIRG

Pleckstrin

homologydo

mainlowast

(A0A

0A6L

5L9)

3333

441651

441487

2900

0plusmn

2700

01600

0plusmn

1100

010

RQHPD

PLSN

QsN

FNsN

TINNYS

NYR

SsTRS

GLD

PsQRH

RhoGTP

asea

ctivating

proteindo

mainlowast

(A0A

0A3B

MU5)

3412

465996

466032

1500

0plusmn

1100

3200plusmn

2700

000

014

KDLP

IGYILH

mIN

LcPN

IVsLNLG

NLSLsTD

YEISRS

TIHKY

F-bo

xproteinCO

S111

(A0A

0A3C

YY6)

3368

465821

KWNKE

KIEL

DsPLIVS

YVSSLC

NGGGGGIIT

NsTNST

ttNSK

Pentatric

opeptid

erepeat

(PPR

)protein

familylowast

(A0A

0A3C

GE7

)111

Misc

ellaneou

s

128483

128542

2700

0plusmn

9500

1400

0plusmn

2800

0016

RNDsD

tsLsK

EInsulin

indu

cedprotein

familylowast

(A0A

0A3C

RM0)

116

6RYtmNEV

FKV

GYF

domainlowast

(A0A

0A3C

0N8)

333128

333159

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

ETsQ

FMGNAES

EDLtGNGLL

TSTL

AVLS

SIS

Orfin

Calbicans

major

repeat

sequ

enceR

B2region

(Q5A

475)

3315

333128

333244

6300

0plusmn

1200

1400

0plusmn

1400

023

KLS

SLGNHGTtTT

SSLS

SSsSsSIsNNT

SIAKI

CBSdo

mainlowast

(A0A

0A6JTB

4)3

346

337660

337659

4300

0plusmn

1200

03300

0plusmn

2800

0RKQ

QDQNEV

AGAAAAT

TTTA

tAtAT

AAT

NWKP

KN

321

349815

349857

3400

0plusmn

8900

1800

0plusmn

1600

00054

KSSsLIK

NsTsSNQSsPA

TSTN

TSIV

DVPIEK


(A0A

0A3C

A48)

3321

417360

417295

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

RIN

NNNDKs

sILsNITTT

NTT

TGTG

TTNTtT

VPS

IKTK

R(A

0A0A

6IY5

3)424

349815

349948

3400

0plusmn

8900

1800

0plusmn

1600

00054

KAFS

SsKL

TSDSA

NStNStNsTSM

SILG

NDKD

CDKinhibitorP

HO81

(A0A

0A6M

Y31)

3316

389536

389486

3600

0plusmn

4100

2600

0plusmn

3100

00037

RISRP

NGVG

GISTS

GSSSP

TTEF

VTP

QAs

KsSV

DQNKK

RUbiqu


(C4Y

NW3)

3329

442103

442109

3200

0plusmn

1900

01700

0plusmn

7200

0080

RHLILG

YKItV

VtDHQSLTsVMTS

SSRP

ENNRM

IRW

Aspartic

peptidasefam

ilylowast

(A0A

0A4C

VF8

)3

383

393914

394016

3600

0plusmn

2400

01700

0plusmn

1800

000026

RNVNGSG

StNtN

TMtRLD

sTTIASSLF

CRQLY

FNLL

SKD

Globinfamilylowast

(A0A

0A4C

D75)

3311

437257

437390

9600plusmn

4600

5400plusmn

1800

0058

RNNsT

VSST

NSL

mSN

NsD

TNtAAT

AAT

AAT

SGS

TTNNVKR

M(A

0A0A

3XE4

6)338


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

441615

441482

2900

0plusmn

2700

01600

0plusmn

1100

010

RNGtYSSsStSSSsTSSVS

SSSA

TTANGES

LNST

THNIQ

LERT

Sec2plowast

(A0A

0A6JGN5)

3289

465996

466133

1500

0plusmn

1100

3200plusmn

2700

000

014

KQNYV

DPG

QIAIKGLK

GFV

NLG

AtCF

MssILQtLIH

NPL

IKY

Ubiqu

itincarboxyl-te

rminal

hydrolase

(C4Y

KS7)

3369

466093

KYD

KPmED

TEEIDDVtSISK

sINEQ

IDDPF

sQFN

SVTL

RY

Protein-serin

ethreon

ine

kinase

(A0A

0A4A

NB2

)35

1

Uncharacterized

117588

117373

6400

0plusmn

4000

2200

0plusmn

1100lt000

001

KtIm

tItIK

I(Q

59T3

9)1

207

333128

333260

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

RIsNIITL

NSSLS

SSsSsSSSsSSLLL

LTL

KS

(Q5A

AP9

)3

311

389536

389370

3600

0plusmn

4100

2600

0plusmn

3100

00034

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

314

393914

394074

3600

0plusmn

2400

01700

0plusmn

1800

000026

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

346

411551

411679

3100

0plusmn

2000

01700

0plusmn

7200

0017

KTV

CNWQtLGHTD

EFEE

stQFA

RGVS

DtAL

VGGITKL

(Q59K0

1)

3

218

411482

TCG

KSCF

INSttANKL

IsYN

LFQSStEVDS

IGPT

GSK

T(Q

59KZ

4)386

41162

KTT

PVMGNSsTP

STVTA

NTN

TGAY

STSsDt

AAKP

TKKA

TKR

(A0A

0A6JKT

0)391

411504

RILLL

LPLL

GVtILTS

KSSLES

ISLN

sPSD

SIALssSKS

(Q5A

H83)

234

441651

441518

2900

0plusmn

2700

01600

0plusmn

1100

010

KST

SSANIKtKPK

PNTA

TtAT

TPTA

tTAT

TtA

TTSSID

PTEK

D(A

0A0A

4CH02)

3259

442103

441999

3200

0plusmn

1900

01700

0plusmn

7200

0080

RLS

GtN

NPG

sGsG

sGGGGGGGANNNSLPG

YSV

GSSIG

RGRG

LGRG

(A0A

0A6LWB8

)3

189

436859

436693

3300

0plusmn

2100

07100plusmn

1300

0020

KNYN

QFK

LIEL

DDSM

NTT

TTTT

TTTT

tTttT

tTST

IGKF

(A0A

0A6M

D82)

3376

436787

KSK

sTTIHNQSN

IHSE

QISIN

DEN

NNKS

tstS

TSTD

TKK

(A0A

0A3E

QM3)

132

437257

437082

9600plusmn

4600

5400plusmn1800

0058

KSSGGSSDTK

SVWIAtTGSD

FASQ

SNSD

SsStASR

NSSSSASR

Q(A

0A0A

3BP13)

3246

437317

KEsEG

VWGWsLQCL

LLLF

TKVNLR

scDQL

LICA

LVIRE

(Q5A

KS1)

299

465996

466136

1500

0plusmn

1100

3200plusmn

2700

000

014

KVILIG

NSM

TsRT

TsSV

RFFSIM

LTVS

LPItN

ILVsSEL

SKV

(A0A

0A3B

Z01)

3311



Biologicalfunction







(A0A0A6JC81)1 182




(A0A0A3BNV6)1 203

DNA binding

57544 57645 9700 plusmn1000



(C4YDL3)1 154

129759

12977518000 plusmn8800

51000 plusmn5400 000042


1

154




156



(A0A0A4CD62) 1 315

190171 190374 7800 plusmn3900


(C4YMX3) 2 319





4 Conclusions


Disclosure



Competing Interests


Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


2 120583m

(a) Control

2 120583m

(b) 30120583gmL

2 120583m

(c) 25120583gmL

2 120583m

(d) 20120583gmL
















ble1Proteins

identifi

edfro

mam

iniaturiz

edassayof

HTN

5tre

atmento

fCalbica

nswith

decreasedMSsig

nals

AverageM

Ssig

nalsandtheirstand

arddeviations

werec

alculatedbased

onsix


icated

with

anasteris

k(lowast)h

aveb

eendeterm

ined


proteinfamiliesLow

ercaselette

rsin

aminoacidsequ

encesind

icated

mod

ified

resid

ues

Blankspaces

indicatedproteins

unidentifi

edby

thed

atabases

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Virulence

128483

128665

2700

0plusmn

9500

1400

0plusmn

2800

0016

KsLYT

ISPN

KGKR

Transcrip

tionalregulator

STP3

(A0A

0A6LWB8

)3

252

128369

KSK

YNLP

FAMKE

Cand

idapepsin

-7(A

0A0A

4B714)

375

129587

129570

6600

0plusmn

1100

1900

0plusmn

7000

000

0013

RtKKW

GLG

WIK

NIntegrasefam

ilylowast

(A0A

0A3B

ML7

)1

161

357979

358147

4000

0plusmn

5000

3100

0plusmn

3600

0030

HPQ

YsEA

CsAV

mVVTY

SSGSG

EHIH

TTDIK

Kexin

(A0A

0A6IYX

0)3

333

389536

389280

3600

0plusmn

4100

2600

0plusmn

3100

00037

ELKT

TVIV

TSCF

NNVC

SETsITTPK

tAVtAT

tSK

Flocculinlowast

(A0A

0A3C

M71)

3310

465996

465822

1500

0plusmn

1100

3200plusmn

2700

000

014

KHLT

LKSSTP

AST

LEYS

TSIPPA

LATT

SSSLStES

TtLttIS

RS

325

411551

411594

5100

0plusmn

1200

02400

0plusmn

1700

00017

VTF

VEK

AtST

STTN

tTttT

TTTT

TTTT

TTT

TIPV


ilylowast

(A0A

0A6K

6B3)

3323

412103

412205

3100

0plusmn

2300

01700

0plusmn

7200

013

KNVKV

ITTT

TTtSPS

SFSSSSSLMsPITPQ

TPN

IPKT

PKT

PXdo

mainlowast

(A0A

0A3C

BE8)

3394

Surfa

ce-

associated

117588

117366

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

RtIN

LDsQ

VKY

31015840(21015840 )51015840-Bisp

hosphate

nucle

otidase2

(A0A

0A6N

TV1)

1316

465996

465987

6600

0plusmn

1100

2000

0plusmn

7000

000

001

mQTS

ISttT

IEDHLH

HYsPE

ESQKL

LSRE

SSIN

TDLF

KE

Budsites

electionprotein

BUD4

(A0A

0A3D

IG6)

3313

Mito

chon

drial-

associated

117588

117515

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KYM

LLTL

LtKL

YAP-bd

ALF

4glom

ulin

familylowast

(A0A

0A3C

AC1)

1296

129587

129768

6600

0plusmn

1100

1900

0plusmn

7000

000

001

KLT

tLISSIEN

KI

296

137401

137670

1600

0plusmn

1300

1000

0plusmn

3000

000

47RTA

SGNIIPS

STGAAKA

Glyceraldehyde-3-ph

osph

ate

dehydrogenase

(Q92211)

1286

465996

466023

1500

0plusmn

1100

3200plusmn

2700

000

014

KStIV

EEIYsN

ARS

HLV

QGNKE

mGmALF

NE

LLAIN

ESIYGKV

Clusteredmito

chon

dria

proteinho

molog

(A0A

0A4C

6G1)

3337

DNAbind

ing

80477

80444

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQPA

VFSRI

DNApo

lymerase

(Q5A

4E5)

1256

128483

128353

2700

0plusmn

9500

1400

0plusmn

2800

0016

RVA

SHsLsTsR

RMinichrom

osom

emaintenance

protein10

(A0A

0A3C

IN8)

1267

333128

333142

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

KGFT

NTM

ISHIG

FDPT

GtsL

NQNsTS

LGsK

S3

321

280676

280620

4700

0plusmn

3600

03000

0plusmn

2300

0021

RTT

PPTV

sITG

PNPsSSPA

sAST

NtSKS

Phosph

atasefam

ilylowast

(A0A

0A3B

QA3)

3360


ble1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

337660

337698

4300

0plusmn

1200

03200

0plusmn

2800

0023

KSSPT

SsAT

TTAT

tSVsISSLSLtMGKP

KNSK

LMediatorc

omplexlowast

(A0A

0A3C

P70)

3315

349815

350080

3400

0plusmn

8900

1800

0plusmn

1600

00054

KLG

SVLT

tRSQ

LIEY

ELtTRtIFIN

CSA

ALK

ISpt6

(D3IZV

2)3

311

350340

350474

4300

0plusmn

8300

3100

0plusmn

2500

0014

REV

IIKL

SITR

tHtPPP

DST

tTTT

PTTS

IEKT

Znfin

gerd

omainlowast

(A0A

0A6J6A

5)3

357

393914

394046

3600

0plusmn

4000

1700

0plusmn

8000

00026

VSSY

ILDGNNST

KLPsPV

LtHtTF

DSR

sDEG

QR

Basic

leucinez

ipperd

omainlowast

(A0A

0A3D

LJ5)

3399

411551

411695

5100

0plusmn

1200

02400

0plusmn

1700

00017

RAPQ

sIQLP

PIQsFtKsQ

AVFP

QSV

RDSA

PAANFN

RY

Transcrip

tionfactor

andDNA

bind


(A0A

0A6ITE

8)3

312

465996

465915

1500

0plusmn

1100

3200plusmn

2700

000

014

MKImmIPTH

HQtYNIN

THQPP

QQHQYL

PPPG

tSY

TSPR

A312

411551

411487

5100

0plusmn

1200

02400

0plusmn

1700

00017

KsStQ

MSSCtNsV

TQTL

DRL

PKIV

STQQNNL

TPTS

KI

Zn(2)-DNAbind

ingdo

mainlowast

(A0A

0A6K

Y81)

3303

465996

466019

1500

0plusmn

1100

3200plusmn

2700

000

014

RRS

VSYS

PGPsSIKS

QLP

HLT

SSST

TtssVQ

SPP

PPPP

SQPP

RG

361

417360

417373

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

KtV

TsIN

GSP

PPLE

tAPsSH

HNVPIDFIHFK

KESD

RT

Uncharacterized

protein

(A0A

0A3E

PD4)

3312

436859

436789

3300

0plusmn

2100

07100plusmn

1300

0020

KNMQFP

PYQVS

sHNsSEtSQ

sIPN

TPSITR

QVE

SNTR

S

Transcrip

tionalregulatory

proteinLE

U3

(A0A

0A3B

TY8)

3354

436790

KtPTT

TTTT

TTtANGNTS

NGNTS

NGNsTGKT

ATA

ATAT

KSNtK

Transcrip


(A0A

0A3C

3Y1)

319

Protein

synthesis

80477

80541

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQTS

LNDKC

Manno

syltransfe

rase

familylowast

(A0A

0A3C

UB4

)1

254

80445

RVKI

DSD

KS

ProteintransporterS

EC24

(A0A

0A3E

HG4)

151

117588

117398

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KTK

QFN

DsK

KK

Vesic

letetheringprotein

familylowast

(A0A

0A6K

IG6)

1201

128483

128372

2700

0plusmn

9500

1400

0plusmn

2800

0016

KtAGsN

HNsEKK

Ribo

somalRN

A-processin

gprotein12

(A0A

0A4B

GJ7)

1251

412103

411993

3100

0plusmn

2300

01700

0plusmn

7200

013

KFF

sDNIANDLA

tTTT

TTTT

TNTG

AtSV

HPIL

QVDAIK

YCA

SCS


(A0A

0A6L

8P2)

3324

436859

437093

3300

0plusmn

2100

07100plusmn

1300

0020

KES

SSTA

DQPS

VVPP

QES

HKD

TVET

PKPE

VtEtsV

EAtKE

Transla


4G(A

0A0A

4CDK4

)3

299

436703

KNPT

PTPT

PTPT

PTPN

NLA

QGVDsSST

LDV

EtTL

tGLtRR

I


sphate

synthase

cycla

sesubu

nit

(A0A

0A6I238)

321


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Sign

allin

g

350340

350460

4300

0plusmn

8300

3100

0plusmn

2500

0013

KRS

SITtPtPP

LTTT

HSSNGNGNGNV

NVNVNsK

R

Peroxisometransmem

brane

receptorlowast

(A0A

0A3Z

WC2

)3

330

411551

411676

3100

0plusmn

2000

01700

0plusmn

7200

0017

KQST

TNTsTL

ssTtAAST

LATS

NNTQ

PDTY

TSTS

TSIRG

Pleckstrin

homologydo

mainlowast

(A0A

0A6L

5L9)

3333

441651

441487

2900

0plusmn

2700

01600

0plusmn

1100

010

RQHPD

PLSN

QsN

FNsN

TINNYS

NYR

SsTRS

GLD

PsQRH

RhoGTP

asea

ctivating

proteindo

mainlowast

(A0A

0A3B

MU5)

3412

465996

466032

1500

0plusmn

1100

3200plusmn

2700

000

014

KDLP

IGYILH

mIN

LcPN

IVsLNLG

NLSLsTD

YEISRS

TIHKY

F-bo

xproteinCO

S111

(A0A

0A3C

YY6)

3368

465821

KWNKE

KIEL

DsPLIVS

YVSSLC

NGGGGGIIT

NsTNST

ttNSK

Pentatric

opeptid

erepeat

(PPR

)protein

familylowast

(A0A

0A3C

GE7

)111

Misc

ellaneou

s

128483

128542

2700

0plusmn

9500

1400

0plusmn

2800

0016

RNDsD

tsLsK

EInsulin

indu

cedprotein

familylowast

(A0A

0A3C

RM0)

116

6RYtmNEV

FKV

GYF

domainlowast

(A0A

0A3C

0N8)

333128

333159

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

ETsQ

FMGNAES

EDLtGNGLL

TSTL

AVLS

SIS

Orfin

Calbicans

major

repeat

sequ

enceR

B2region

(Q5A

475)

3315

333128

333244

6300

0plusmn

1200

1400

0plusmn

1400

023

KLS

SLGNHGTtTT

SSLS

SSsSsSIsNNT

SIAKI

CBSdo

mainlowast

(A0A

0A6JTB

4)3

346

337660

337659

4300

0plusmn

1200

03300

0plusmn

2800

0RKQ

QDQNEV

AGAAAAT

TTTA

tAtAT

AAT

NWKP

KN

321

349815

349857

3400

0plusmn

8900

1800

0plusmn

1600

00054

KSSsLIK

NsTsSNQSsPA

TSTN

TSIV

DVPIEK


(A0A

0A3C

A48)

3321

417360

417295

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

RIN

NNNDKs

sILsNITTT

NTT

TGTG

TTNTtT

VPS

IKTK

R(A

0A0A

6IY5

3)424

349815

349948

3400

0plusmn

8900

1800

0plusmn

1600

00054

KAFS

SsKL

TSDSA

NStNStNsTSM

SILG

NDKD

CDKinhibitorP

HO81

(A0A

0A6M

Y31)

3316

389536

389486

3600

0plusmn

4100

2600

0plusmn

3100

00037

RISRP

NGVG

GISTS

GSSSP

TTEF

VTP

QAs

KsSV

DQNKK

RUbiqu


(C4Y

NW3)

3329

442103

442109

3200

0plusmn

1900

01700

0plusmn

7200

0080

RHLILG

YKItV

VtDHQSLTsVMTS

SSRP

ENNRM

IRW

Aspartic

peptidasefam

ilylowast

(A0A

0A4C

VF8

)3

383

393914

394016

3600

0plusmn

2400

01700

0plusmn

1800

000026

RNVNGSG

StNtN

TMtRLD

sTTIASSLF

CRQLY

FNLL

SKD

Globinfamilylowast

(A0A

0A4C

D75)

3311

437257

437390

9600plusmn

4600

5400plusmn

1800

0058

RNNsT

VSST

NSL

mSN

NsD

TNtAAT

AAT

AAT

SGS

TTNNVKR

M(A

0A0A

3XE4

6)338


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

441615

441482

2900

0plusmn

2700

01600

0plusmn

1100

010

RNGtYSSsStSSSsTSSVS

SSSA

TTANGES

LNST

THNIQ

LERT

Sec2plowast

(A0A

0A6JGN5)

3289

465996

466133

1500

0plusmn

1100

3200plusmn

2700

000

014

KQNYV

DPG

QIAIKGLK

GFV

NLG

AtCF

MssILQtLIH

NPL

IKY

Ubiqu

itincarboxyl-te

rminal

hydrolase

(C4Y

KS7)

3369

466093

KYD

KPmED

TEEIDDVtSISK

sINEQ

IDDPF

sQFN

SVTL

RY

Protein-serin

ethreon

ine

kinase

(A0A

0A4A

NB2

)35

1

Uncharacterized

117588

117373

6400

0plusmn

4000

2200

0plusmn

1100lt000

001

KtIm

tItIK

I(Q

59T3

9)1

207

333128

333260

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

RIsNIITL

NSSLS

SSsSsSSSsSSLLL

LTL

KS

(Q5A

AP9

)3

311

389536

389370

3600

0plusmn

4100

2600

0plusmn

3100

00034

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

314

393914

394074

3600

0plusmn

2400

01700

0plusmn

1800

000026

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

346

411551

411679

3100

0plusmn

2000

01700

0plusmn

7200

0017

KTV

CNWQtLGHTD

EFEE

stQFA

RGVS

DtAL

VGGITKL

(Q59K0

1)

3

218

411482

TCG

KSCF

INSttANKL

IsYN

LFQSStEVDS

IGPT

GSK

T(Q

59KZ

4)386

41162

KTT

PVMGNSsTP

STVTA

NTN

TGAY

STSsDt

AAKP

TKKA

TKR

(A0A

0A6JKT

0)391

411504

RILLL

LPLL

GVtILTS

KSSLES

ISLN

sPSD

SIALssSKS

(Q5A

H83)

234

441651

441518

2900

0plusmn

2700

01600

0plusmn

1100

010

KST

SSANIKtKPK

PNTA

TtAT

TPTA

tTAT

TtA

TTSSID

PTEK

D(A

0A0A

4CH02)

3259

442103

441999

3200

0plusmn

1900

01700

0plusmn

7200

0080

RLS

GtN

NPG

sGsG

sGGGGGGGANNNSLPG

YSV

GSSIG

RGRG

LGRG

(A0A

0A6LWB8

)3

189

436859

436693

3300

0plusmn

2100

07100plusmn

1300

0020

KNYN

QFK

LIEL

DDSM

NTT

TTTT

TTTT

tTttT

tTST

IGKF

(A0A

0A6M

D82)

3376

436787

KSK

sTTIHNQSN

IHSE

QISIN

DEN

NNKS

tstS

TSTD

TKK

(A0A

0A3E

QM3)

132

437257

437082

9600plusmn

4600

5400plusmn1800

0058

KSSGGSSDTK

SVWIAtTGSD

FASQ

SNSD

SsStASR

NSSSSASR

Q(A

0A0A

3BP13)

3246

437317

KEsEG

VWGWsLQCL

LLLF

TKVNLR

scDQL

LICA

LVIRE

(Q5A

KS1)

299

465996

466136

1500

0plusmn

1100

3200plusmn

2700

000

014

KVILIG

NSM

TsRT

TsSV

RFFSIM

LTVS

LPItN

ILVsSEL

SKV

(A0A

0A3B

Z01)

3311



Biologicalfunction







(A0A0A6JC81)1 182




(A0A0A3BNV6)1 203

DNA binding

57544 57645 9700 plusmn1000



(C4YDL3)1 154

129759

12977518000 plusmn8800

51000 plusmn5400 000042


1

154




156



(A0A0A4CD62) 1 315

190171 190374 7800 plusmn3900


(C4YMX3) 2 319





4 Conclusions


Disclosure



Competing Interests


Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










ble1Proteins

identifi

edfro

mam

iniaturiz

edassayof

HTN

5tre

atmento

fCalbica

nswith

decreasedMSsig

nals

AverageM

Ssig

nalsandtheirstand

arddeviations

werec

alculatedbased

onsix


icated

with

anasteris

k(lowast)h

aveb

eendeterm

ined


proteinfamiliesLow

ercaselette

rsin

aminoacidsequ

encesind

icated

mod

ified

resid

ues

Blankspaces

indicatedproteins

unidentifi

edby

thed

atabases

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Virulence

128483

128665

2700

0plusmn

9500

1400

0plusmn

2800

0016

KsLYT

ISPN

KGKR

Transcrip

tionalregulator

STP3

(A0A

0A6LWB8

)3

252

128369

KSK

YNLP

FAMKE

Cand

idapepsin

-7(A

0A0A

4B714)

375

129587

129570

6600

0plusmn

1100

1900

0plusmn

7000

000

0013

RtKKW

GLG

WIK

NIntegrasefam

ilylowast

(A0A

0A3B

ML7

)1

161

357979

358147

4000

0plusmn

5000

3100

0plusmn

3600

0030

HPQ

YsEA

CsAV

mVVTY

SSGSG

EHIH

TTDIK

Kexin

(A0A

0A6IYX

0)3

333

389536

389280

3600

0plusmn

4100

2600

0plusmn

3100

00037

ELKT

TVIV

TSCF

NNVC

SETsITTPK

tAVtAT

tSK

Flocculinlowast

(A0A

0A3C

M71)

3310

465996

465822

1500

0plusmn

1100

3200plusmn

2700

000

014

KHLT

LKSSTP

AST

LEYS

TSIPPA

LATT

SSSLStES

TtLttIS

RS

325

411551

411594

5100

0plusmn

1200

02400

0plusmn

1700

00017

VTF

VEK

AtST

STTN

tTttT

TTTT

TTTT

TTT

TIPV


ilylowast

(A0A

0A6K

6B3)

3323

412103

412205

3100

0plusmn

2300

01700

0plusmn

7200

013

KNVKV

ITTT

TTtSPS

SFSSSSSLMsPITPQ

TPN

IPKT

PKT

PXdo

mainlowast

(A0A

0A3C

BE8)

3394

Surfa

ce-

associated

117588

117366

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

RtIN

LDsQ

VKY

31015840(21015840 )51015840-Bisp

hosphate

nucle

otidase2

(A0A

0A6N

TV1)

1316

465996

465987

6600

0plusmn

1100

2000

0plusmn

7000

000

001

mQTS

ISttT

IEDHLH

HYsPE

ESQKL

LSRE

SSIN

TDLF

KE

Budsites

electionprotein

BUD4

(A0A

0A3D

IG6)

3313

Mito

chon

drial-

associated

117588

117515

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KYM

LLTL

LtKL

YAP-bd

ALF

4glom

ulin

familylowast

(A0A

0A3C

AC1)

1296

129587

129768

6600

0plusmn

1100

1900

0plusmn

7000

000

001

KLT

tLISSIEN

KI

296

137401

137670

1600

0plusmn

1300

1000

0plusmn

3000

000

47RTA

SGNIIPS

STGAAKA

Glyceraldehyde-3-ph

osph

ate

dehydrogenase

(Q92211)

1286

465996

466023

1500

0plusmn

1100

3200plusmn

2700

000

014

KStIV

EEIYsN

ARS

HLV

QGNKE

mGmALF

NE

LLAIN

ESIYGKV

Clusteredmito

chon

dria

proteinho

molog

(A0A

0A4C

6G1)

3337

DNAbind

ing

80477

80444

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQPA

VFSRI

DNApo

lymerase

(Q5A

4E5)

1256

128483

128353

2700

0plusmn

9500

1400

0plusmn

2800

0016

RVA

SHsLsTsR

RMinichrom

osom

emaintenance

protein10

(A0A

0A3C

IN8)

1267

333128

333142

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

KGFT

NTM

ISHIG

FDPT

GtsL

NQNsTS

LGsK

S3

321

280676

280620

4700

0plusmn

3600

03000

0plusmn

2300

0021

RTT

PPTV

sITG

PNPsSSPA

sAST

NtSKS

Phosph

atasefam

ilylowast

(A0A

0A3B

QA3)

3360


ble1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

337660

337698

4300

0plusmn

1200

03200

0plusmn

2800

0023

KSSPT

SsAT

TTAT

tSVsISSLSLtMGKP

KNSK

LMediatorc

omplexlowast

(A0A

0A3C

P70)

3315

349815

350080

3400

0plusmn

8900

1800

0plusmn

1600

00054

KLG

SVLT

tRSQ

LIEY

ELtTRtIFIN

CSA

ALK

ISpt6

(D3IZV

2)3

311

350340

350474

4300

0plusmn

8300

3100

0plusmn

2500

0014

REV

IIKL

SITR

tHtPPP

DST

tTTT

PTTS

IEKT

Znfin

gerd

omainlowast

(A0A

0A6J6A

5)3

357

393914

394046

3600

0plusmn

4000

1700

0plusmn

8000

00026

VSSY

ILDGNNST

KLPsPV

LtHtTF

DSR

sDEG

QR

Basic

leucinez

ipperd

omainlowast

(A0A

0A3D

LJ5)

3399

411551

411695

5100

0plusmn

1200

02400

0plusmn

1700

00017

RAPQ

sIQLP

PIQsFtKsQ

AVFP

QSV

RDSA

PAANFN

RY

Transcrip

tionfactor

andDNA

bind


(A0A

0A6ITE

8)3

312

465996

465915

1500

0plusmn

1100

3200plusmn

2700

000

014

MKImmIPTH

HQtYNIN

THQPP

QQHQYL

PPPG

tSY

TSPR

A312

411551

411487

5100

0plusmn

1200

02400

0plusmn

1700

00017

KsStQ

MSSCtNsV

TQTL

DRL

PKIV

STQQNNL

TPTS

KI

Zn(2)-DNAbind

ingdo

mainlowast

(A0A

0A6K

Y81)

3303

465996

466019

1500

0plusmn

1100

3200plusmn

2700

000

014

RRS

VSYS

PGPsSIKS

QLP

HLT

SSST

TtssVQ

SPP

PPPP

SQPP

RG

361

417360

417373

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

KtV

TsIN

GSP

PPLE

tAPsSH

HNVPIDFIHFK

KESD

RT

Uncharacterized

protein

(A0A

0A3E

PD4)

3312

436859

436789

3300

0plusmn

2100

07100plusmn

1300

0020

KNMQFP

PYQVS

sHNsSEtSQ

sIPN

TPSITR

QVE

SNTR

S

Transcrip

tionalregulatory

proteinLE

U3

(A0A

0A3B

TY8)

3354

436790

KtPTT

TTTT

TTtANGNTS

NGNTS

NGNsTGKT

ATA

ATAT

KSNtK

Transcrip


(A0A

0A3C

3Y1)

319

Protein

synthesis

80477

80541

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQTS

LNDKC

Manno

syltransfe

rase

familylowast

(A0A

0A3C

UB4

)1

254

80445

RVKI

DSD

KS

ProteintransporterS

EC24

(A0A

0A3E

HG4)

151

117588

117398

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KTK

QFN

DsK

KK

Vesic

letetheringprotein

familylowast

(A0A

0A6K

IG6)

1201

128483

128372

2700

0plusmn

9500

1400

0plusmn

2800

0016

KtAGsN

HNsEKK

Ribo

somalRN

A-processin

gprotein12

(A0A

0A4B

GJ7)

1251

412103

411993

3100

0plusmn

2300

01700

0plusmn

7200

013

KFF

sDNIANDLA

tTTT

TTTT

TNTG

AtSV

HPIL

QVDAIK

YCA

SCS


(A0A

0A6L

8P2)

3324

436859

437093

3300

0plusmn

2100

07100plusmn

1300

0020

KES

SSTA

DQPS

VVPP

QES

HKD

TVET

PKPE

VtEtsV

EAtKE

Transla


4G(A

0A0A

4CDK4

)3

299

436703

KNPT

PTPT

PTPT

PTPN

NLA

QGVDsSST

LDV

EtTL

tGLtRR

I


sphate

synthase

cycla

sesubu

nit

(A0A

0A6I238)

321


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Sign

allin

g

350340

350460

4300

0plusmn

8300

3100

0plusmn

2500

0013

KRS

SITtPtPP

LTTT

HSSNGNGNGNV

NVNVNsK

R

Peroxisometransmem

brane

receptorlowast

(A0A

0A3Z

WC2

)3

330

411551

411676

3100

0plusmn

2000

01700

0plusmn

7200

0017

KQST

TNTsTL

ssTtAAST

LATS

NNTQ

PDTY

TSTS

TSIRG

Pleckstrin

homologydo

mainlowast

(A0A

0A6L

5L9)

3333

441651

441487

2900

0plusmn

2700

01600

0plusmn

1100

010

RQHPD

PLSN

QsN

FNsN

TINNYS

NYR

SsTRS

GLD

PsQRH

RhoGTP

asea

ctivating

proteindo

mainlowast

(A0A

0A3B

MU5)

3412

465996

466032

1500

0plusmn

1100

3200plusmn

2700

000

014

KDLP

IGYILH

mIN

LcPN

IVsLNLG

NLSLsTD

YEISRS

TIHKY

F-bo

xproteinCO

S111

(A0A

0A3C

YY6)

3368

465821

KWNKE

KIEL

DsPLIVS

YVSSLC

NGGGGGIIT

NsTNST

ttNSK

Pentatric

opeptid

erepeat

(PPR

)protein

familylowast

(A0A

0A3C

GE7

)111

Misc

ellaneou

s

128483

128542

2700

0plusmn

9500

1400

0plusmn

2800

0016

RNDsD

tsLsK

EInsulin

indu

cedprotein

familylowast

(A0A

0A3C

RM0)

116

6RYtmNEV

FKV

GYF

domainlowast

(A0A

0A3C

0N8)

333128

333159

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

ETsQ

FMGNAES

EDLtGNGLL

TSTL

AVLS

SIS

Orfin

Calbicans

major

repeat

sequ

enceR

B2region

(Q5A

475)

3315

333128

333244

6300

0plusmn

1200

1400

0plusmn

1400

023

KLS

SLGNHGTtTT

SSLS

SSsSsSIsNNT

SIAKI

CBSdo

mainlowast

(A0A

0A6JTB

4)3

346

337660

337659

4300

0plusmn

1200

03300

0plusmn

2800

0RKQ

QDQNEV

AGAAAAT

TTTA

tAtAT

AAT

NWKP

KN

321

349815

349857

3400

0plusmn

8900

1800

0plusmn

1600

00054

KSSsLIK

NsTsSNQSsPA

TSTN

TSIV

DVPIEK


(A0A

0A3C

A48)

3321

417360

417295

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

RIN

NNNDKs

sILsNITTT

NTT

TGTG

TTNTtT

VPS

IKTK

R(A

0A0A

6IY5

3)424

349815

349948

3400

0plusmn

8900

1800

0plusmn

1600

00054

KAFS

SsKL

TSDSA

NStNStNsTSM

SILG

NDKD

CDKinhibitorP

HO81

(A0A

0A6M

Y31)

3316

389536

389486

3600

0plusmn

4100

2600

0plusmn

3100

00037

RISRP

NGVG

GISTS

GSSSP

TTEF

VTP

QAs

KsSV

DQNKK

RUbiqu


(C4Y

NW3)

3329

442103

442109

3200

0plusmn

1900

01700

0plusmn

7200

0080

RHLILG

YKItV

VtDHQSLTsVMTS

SSRP

ENNRM

IRW

Aspartic

peptidasefam

ilylowast

(A0A

0A4C

VF8

)3

383

393914

394016

3600

0plusmn

2400

01700

0plusmn

1800

000026

RNVNGSG

StNtN

TMtRLD

sTTIASSLF

CRQLY

FNLL

SKD

Globinfamilylowast

(A0A

0A4C

D75)

3311

437257

437390

9600plusmn

4600

5400plusmn

1800

0058

RNNsT

VSST

NSL

mSN

NsD

TNtAAT

AAT

AAT

SGS

TTNNVKR

M(A

0A0A

3XE4

6)338


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

441615

441482

2900

0plusmn

2700

01600

0plusmn

1100

010

RNGtYSSsStSSSsTSSVS

SSSA

TTANGES

LNST

THNIQ

LERT

Sec2plowast

(A0A

0A6JGN5)

3289

465996

466133

1500

0plusmn

1100

3200plusmn

2700

000

014

KQNYV

DPG

QIAIKGLK

GFV

NLG

AtCF

MssILQtLIH

NPL

IKY

Ubiqu

itincarboxyl-te

rminal

hydrolase

(C4Y

KS7)

3369

466093

KYD

KPmED

TEEIDDVtSISK

sINEQ

IDDPF

sQFN

SVTL

RY

Protein-serin

ethreon

ine

kinase

(A0A

0A4A

NB2

)35

1

Uncharacterized

117588

117373

6400

0plusmn

4000

2200

0plusmn

1100lt000

001

KtIm

tItIK

I(Q

59T3

9)1

207

333128

333260

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

RIsNIITL

NSSLS

SSsSsSSSsSSLLL

LTL

KS

(Q5A

AP9

)3

311

389536

389370

3600

0plusmn

4100

2600

0plusmn

3100

00034

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

314

393914

394074

3600

0plusmn

2400

01700

0plusmn

1800

000026

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

346

411551

411679

3100

0plusmn

2000

01700

0plusmn

7200

0017

KTV

CNWQtLGHTD

EFEE

stQFA

RGVS

DtAL

VGGITKL

(Q59K0

1)

3

218

411482

TCG

KSCF

INSttANKL

IsYN

LFQSStEVDS

IGPT

GSK

T(Q

59KZ

4)386

41162

KTT

PVMGNSsTP

STVTA

NTN

TGAY

STSsDt

AAKP

TKKA

TKR

(A0A

0A6JKT

0)391

411504

RILLL

LPLL

GVtILTS

KSSLES

ISLN

sPSD

SIALssSKS

(Q5A

H83)

234

441651

441518

2900

0plusmn

2700

01600

0plusmn

1100

010

KST

SSANIKtKPK

PNTA

TtAT

TPTA

tTAT

TtA

TTSSID

PTEK

D(A

0A0A

4CH02)

3259

442103

441999

3200

0plusmn

1900

01700

0plusmn

7200

0080

RLS

GtN

NPG

sGsG

sGGGGGGGANNNSLPG

YSV

GSSIG

RGRG

LGRG

(A0A

0A6LWB8

)3

189

436859

436693

3300

0plusmn

2100

07100plusmn

1300

0020

KNYN

QFK

LIEL

DDSM

NTT

TTTT

TTTT

tTttT

tTST

IGKF

(A0A

0A6M

D82)

3376

436787

KSK

sTTIHNQSN

IHSE

QISIN

DEN

NNKS

tstS

TSTD

TKK

(A0A

0A3E

QM3)

132

437257

437082

9600plusmn

4600

5400plusmn1800

0058

KSSGGSSDTK

SVWIAtTGSD

FASQ

SNSD

SsStASR

NSSSSASR

Q(A

0A0A

3BP13)

3246

437317

KEsEG

VWGWsLQCL

LLLF

TKVNLR

scDQL

LICA

LVIRE

(Q5A

KS1)

299

465996

466136

1500

0plusmn

1100

3200plusmn

2700

000

014

KVILIG

NSM

TsRT

TsSV

RFFSIM

LTVS

LPItN

ILVsSEL

SKV

(A0A

0A3B

Z01)

3311



Biologicalfunction







(A0A0A6JC81)1 182




(A0A0A3BNV6)1 203

DNA binding

57544 57645 9700 plusmn1000



(C4YDL3)1 154

129759

12977518000 plusmn8800

51000 plusmn5400 000042


1

154




156



(A0A0A4CD62) 1 315

190171 190374 7800 plusmn3900


(C4YMX3) 2 319





4 Conclusions


Disclosure



Competing Interests


Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


ble1Proteins

identifi

edfro

mam

iniaturiz

edassayof

HTN

5tre

atmento

fCalbica

nswith

decreasedMSsig

nals

AverageM

Ssig

nalsandtheirstand

arddeviations

werec

alculatedbased

onsix


icated

with

anasteris

k(lowast)h

aveb

eendeterm

ined


proteinfamiliesLow

ercaselette

rsin

aminoacidsequ

encesind

icated

mod

ified

resid

ues

Blankspaces

indicatedproteins

unidentifi

edby

thed

atabases

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Virulence

128483

128665

2700

0plusmn

9500

1400

0plusmn

2800

0016

KsLYT

ISPN

KGKR

Transcrip

tionalregulator

STP3

(A0A

0A6LWB8

)3

252

128369

KSK

YNLP

FAMKE

Cand

idapepsin

-7(A

0A0A

4B714)

375

129587

129570

6600

0plusmn

1100

1900

0plusmn

7000

000

0013

RtKKW

GLG

WIK

NIntegrasefam

ilylowast

(A0A

0A3B

ML7

)1

161

357979

358147

4000

0plusmn

5000

3100

0plusmn

3600

0030

HPQ

YsEA

CsAV

mVVTY

SSGSG

EHIH

TTDIK

Kexin

(A0A

0A6IYX

0)3

333

389536

389280

3600

0plusmn

4100

2600

0plusmn

3100

00037

ELKT

TVIV

TSCF

NNVC

SETsITTPK

tAVtAT

tSK

Flocculinlowast

(A0A

0A3C

M71)

3310

465996

465822

1500

0plusmn

1100

3200plusmn

2700

000

014

KHLT

LKSSTP

AST

LEYS

TSIPPA

LATT

SSSLStES

TtLttIS

RS

325

411551

411594

5100

0plusmn

1200

02400

0plusmn

1700

00017

VTF

VEK

AtST

STTN

tTttT

TTTT

TTTT

TTT

TIPV


ilylowast

(A0A

0A6K

6B3)

3323

412103

412205

3100

0plusmn

2300

01700

0plusmn

7200

013

KNVKV

ITTT

TTtSPS

SFSSSSSLMsPITPQ

TPN

IPKT

PKT

PXdo

mainlowast

(A0A

0A3C

BE8)

3394

Surfa

ce-

associated

117588

117366

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

RtIN

LDsQ

VKY

31015840(21015840 )51015840-Bisp

hosphate

nucle

otidase2

(A0A

0A6N

TV1)

1316

465996

465987

6600

0plusmn

1100

2000

0plusmn

7000

000

001

mQTS

ISttT

IEDHLH

HYsPE

ESQKL

LSRE

SSIN

TDLF

KE

Budsites

electionprotein

BUD4

(A0A

0A3D

IG6)

3313

Mito

chon

drial-

associated

117588

117515

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KYM

LLTL

LtKL

YAP-bd

ALF

4glom

ulin

familylowast

(A0A

0A3C

AC1)

1296

129587

129768

6600

0plusmn

1100

1900

0plusmn

7000

000

001

KLT

tLISSIEN

KI

296

137401

137670

1600

0plusmn

1300

1000

0plusmn

3000

000

47RTA

SGNIIPS

STGAAKA

Glyceraldehyde-3-ph

osph

ate

dehydrogenase

(Q92211)

1286

465996

466023

1500

0plusmn

1100

3200plusmn

2700

000

014

KStIV

EEIYsN

ARS

HLV

QGNKE

mGmALF

NE

LLAIN

ESIYGKV

Clusteredmito

chon

dria

proteinho

molog

(A0A

0A4C

6G1)

3337

DNAbind

ing

80477

80444

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQPA

VFSRI

DNApo

lymerase

(Q5A

4E5)

1256

128483

128353

2700

0plusmn

9500

1400

0plusmn

2800

0016

RVA

SHsLsTsR

RMinichrom

osom

emaintenance

protein10

(A0A

0A3C

IN8)

1267

333128

333142

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

KGFT

NTM

ISHIG

FDPT

GtsL

NQNsTS

LGsK

S3

321

280676

280620

4700

0plusmn

3600

03000

0plusmn

2300

0021

RTT

PPTV

sITG

PNPsSSPA

sAST

NtSKS

Phosph

atasefam

ilylowast

(A0A

0A3B

QA3)

3360


ble1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

337660

337698

4300

0plusmn

1200

03200

0plusmn

2800

0023

KSSPT

SsAT

TTAT

tSVsISSLSLtMGKP

KNSK

LMediatorc

omplexlowast

(A0A

0A3C

P70)

3315

349815

350080

3400

0plusmn

8900

1800

0plusmn

1600

00054

KLG

SVLT

tRSQ

LIEY

ELtTRtIFIN

CSA

ALK

ISpt6

(D3IZV

2)3

311

350340

350474

4300

0plusmn

8300

3100

0plusmn

2500

0014

REV

IIKL

SITR

tHtPPP

DST

tTTT

PTTS

IEKT

Znfin

gerd

omainlowast

(A0A

0A6J6A

5)3

357

393914

394046

3600

0plusmn

4000

1700

0plusmn

8000

00026

VSSY

ILDGNNST

KLPsPV

LtHtTF

DSR

sDEG

QR

Basic

leucinez

ipperd

omainlowast

(A0A

0A3D

LJ5)

3399

411551

411695

5100

0plusmn

1200

02400

0plusmn

1700

00017

RAPQ

sIQLP

PIQsFtKsQ

AVFP

QSV

RDSA

PAANFN

RY

Transcrip

tionfactor

andDNA

bind


(A0A

0A6ITE

8)3

312

465996

465915

1500

0plusmn

1100

3200plusmn

2700

000

014

MKImmIPTH

HQtYNIN

THQPP

QQHQYL

PPPG

tSY

TSPR

A312

411551

411487

5100

0plusmn

1200

02400

0plusmn

1700

00017

KsStQ

MSSCtNsV

TQTL

DRL

PKIV

STQQNNL

TPTS

KI

Zn(2)-DNAbind

ingdo

mainlowast

(A0A

0A6K

Y81)

3303

465996

466019

1500

0plusmn

1100

3200plusmn

2700

000

014

RRS

VSYS

PGPsSIKS

QLP

HLT

SSST

TtssVQ

SPP

PPPP

SQPP

RG

361

417360

417373

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

KtV

TsIN

GSP

PPLE

tAPsSH

HNVPIDFIHFK

KESD

RT

Uncharacterized

protein

(A0A

0A3E

PD4)

3312

436859

436789

3300

0plusmn

2100

07100plusmn

1300

0020

KNMQFP

PYQVS

sHNsSEtSQ

sIPN

TPSITR

QVE

SNTR

S

Transcrip

tionalregulatory

proteinLE

U3

(A0A

0A3B

TY8)

3354

436790

KtPTT

TTTT

TTtANGNTS

NGNTS

NGNsTGKT

ATA

ATAT

KSNtK

Transcrip


(A0A

0A3C

3Y1)

319

Protein

synthesis

80477

80541

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQTS

LNDKC

Manno

syltransfe

rase

familylowast

(A0A

0A3C

UB4

)1

254

80445

RVKI

DSD

KS

ProteintransporterS

EC24

(A0A

0A3E

HG4)

151

117588

117398

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KTK

QFN

DsK

KK

Vesic

letetheringprotein

familylowast

(A0A

0A6K

IG6)

1201

128483

128372

2700

0plusmn

9500

1400

0plusmn

2800

0016

KtAGsN

HNsEKK

Ribo

somalRN

A-processin

gprotein12

(A0A

0A4B

GJ7)

1251

412103

411993

3100

0plusmn

2300

01700

0plusmn

7200

013

KFF

sDNIANDLA

tTTT

TTTT

TNTG

AtSV

HPIL

QVDAIK

YCA

SCS


(A0A

0A6L

8P2)

3324

436859

437093

3300

0plusmn

2100

07100plusmn

1300

0020

KES

SSTA

DQPS

VVPP

QES

HKD

TVET

PKPE

VtEtsV

EAtKE

Transla


4G(A

0A0A

4CDK4

)3

299

436703

KNPT

PTPT

PTPT

PTPN

NLA

QGVDsSST

LDV

EtTL

tGLtRR

I


sphate

synthase

cycla

sesubu

nit

(A0A

0A6I238)

321


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Sign

allin

g

350340

350460

4300

0plusmn

8300

3100

0plusmn

2500

0013

KRS

SITtPtPP

LTTT

HSSNGNGNGNV

NVNVNsK

R

Peroxisometransmem

brane

receptorlowast

(A0A

0A3Z

WC2

)3

330

411551

411676

3100

0plusmn

2000

01700

0plusmn

7200

0017

KQST

TNTsTL

ssTtAAST

LATS

NNTQ

PDTY

TSTS

TSIRG

Pleckstrin

homologydo

mainlowast

(A0A

0A6L

5L9)

3333

441651

441487

2900

0plusmn

2700

01600

0plusmn

1100

010

RQHPD

PLSN

QsN

FNsN

TINNYS

NYR

SsTRS

GLD

PsQRH

RhoGTP

asea

ctivating

proteindo

mainlowast

(A0A

0A3B

MU5)

3412

465996

466032

1500

0plusmn

1100

3200plusmn

2700

000

014

KDLP

IGYILH

mIN

LcPN

IVsLNLG

NLSLsTD

YEISRS

TIHKY

F-bo

xproteinCO

S111

(A0A

0A3C

YY6)

3368

465821

KWNKE

KIEL

DsPLIVS

YVSSLC

NGGGGGIIT

NsTNST

ttNSK

Pentatric

opeptid

erepeat

(PPR

)protein

familylowast

(A0A

0A3C

GE7

)111

Misc

ellaneou

s

128483

128542

2700

0plusmn

9500

1400

0plusmn

2800

0016

RNDsD

tsLsK

EInsulin

indu

cedprotein

familylowast

(A0A

0A3C

RM0)

116

6RYtmNEV

FKV

GYF

domainlowast

(A0A

0A3C

0N8)

333128

333159

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

ETsQ

FMGNAES

EDLtGNGLL

TSTL

AVLS

SIS

Orfin

Calbicans

major

repeat

sequ

enceR

B2region

(Q5A

475)

3315

333128

333244

6300

0plusmn

1200

1400

0plusmn

1400

023

KLS

SLGNHGTtTT

SSLS

SSsSsSIsNNT

SIAKI

CBSdo

mainlowast

(A0A

0A6JTB

4)3

346

337660

337659

4300

0plusmn

1200

03300

0plusmn

2800

0RKQ

QDQNEV

AGAAAAT

TTTA

tAtAT

AAT

NWKP

KN

321

349815

349857

3400

0plusmn

8900

1800

0plusmn

1600

00054

KSSsLIK

NsTsSNQSsPA

TSTN

TSIV

DVPIEK


(A0A

0A3C

A48)

3321

417360

417295

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

RIN

NNNDKs

sILsNITTT

NTT

TGTG

TTNTtT

VPS

IKTK

R(A

0A0A

6IY5

3)424

349815

349948

3400

0plusmn

8900

1800

0plusmn

1600

00054

KAFS

SsKL

TSDSA

NStNStNsTSM

SILG

NDKD

CDKinhibitorP

HO81

(A0A

0A6M

Y31)

3316

389536

389486

3600

0plusmn

4100

2600

0plusmn

3100

00037

RISRP

NGVG

GISTS

GSSSP

TTEF

VTP

QAs

KsSV

DQNKK

RUbiqu


(C4Y

NW3)

3329

442103

442109

3200

0plusmn

1900

01700

0plusmn

7200

0080

RHLILG

YKItV

VtDHQSLTsVMTS

SSRP

ENNRM

IRW

Aspartic

peptidasefam

ilylowast

(A0A

0A4C

VF8

)3

383

393914

394016

3600

0plusmn

2400

01700

0plusmn

1800

000026

RNVNGSG

StNtN

TMtRLD

sTTIASSLF

CRQLY

FNLL

SKD

Globinfamilylowast

(A0A

0A4C

D75)

3311

437257

437390

9600plusmn

4600

5400plusmn

1800

0058

RNNsT

VSST

NSL

mSN

NsD

TNtAAT

AAT

AAT

SGS

TTNNVKR

M(A

0A0A

3XE4

6)338


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

441615

441482

2900

0plusmn

2700

01600

0plusmn

1100

010

RNGtYSSsStSSSsTSSVS

SSSA

TTANGES

LNST

THNIQ

LERT

Sec2plowast

(A0A

0A6JGN5)

3289

465996

466133

1500

0plusmn

1100

3200plusmn

2700

000

014

KQNYV

DPG

QIAIKGLK

GFV

NLG

AtCF

MssILQtLIH

NPL

IKY

Ubiqu

itincarboxyl-te

rminal

hydrolase

(C4Y

KS7)

3369

466093

KYD

KPmED

TEEIDDVtSISK

sINEQ

IDDPF

sQFN

SVTL

RY

Protein-serin

ethreon

ine

kinase

(A0A

0A4A

NB2

)35

1

Uncharacterized

117588

117373

6400

0plusmn

4000

2200

0plusmn

1100lt000

001

KtIm

tItIK

I(Q

59T3

9)1

207

333128

333260

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

RIsNIITL

NSSLS

SSsSsSSSsSSLLL

LTL

KS

(Q5A

AP9

)3

311

389536

389370

3600

0plusmn

4100

2600

0plusmn

3100

00034

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

314

393914

394074

3600

0plusmn

2400

01700

0plusmn

1800

000026

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

346

411551

411679

3100

0plusmn

2000

01700

0plusmn

7200

0017

KTV

CNWQtLGHTD

EFEE

stQFA

RGVS

DtAL

VGGITKL

(Q59K0

1)

3

218

411482

TCG

KSCF

INSttANKL

IsYN

LFQSStEVDS

IGPT

GSK

T(Q

59KZ

4)386

41162

KTT

PVMGNSsTP

STVTA

NTN

TGAY

STSsDt

AAKP

TKKA

TKR

(A0A

0A6JKT

0)391

411504

RILLL

LPLL

GVtILTS

KSSLES

ISLN

sPSD

SIALssSKS

(Q5A

H83)

234

441651

441518

2900

0plusmn

2700

01600

0plusmn

1100

010

KST

SSANIKtKPK

PNTA

TtAT

TPTA

tTAT

TtA

TTSSID

PTEK

D(A

0A0A

4CH02)

3259

442103

441999

3200

0plusmn

1900

01700

0plusmn

7200

0080

RLS

GtN

NPG

sGsG

sGGGGGGGANNNSLPG

YSV

GSSIG

RGRG

LGRG

(A0A

0A6LWB8

)3

189

436859

436693

3300

0plusmn

2100

07100plusmn

1300

0020

KNYN

QFK

LIEL

DDSM

NTT

TTTT

TTTT

tTttT

tTST

IGKF

(A0A

0A6M

D82)

3376

436787

KSK

sTTIHNQSN

IHSE

QISIN

DEN

NNKS

tstS

TSTD

TKK

(A0A

0A3E

QM3)

132

437257

437082

9600plusmn

4600

5400plusmn1800

0058

KSSGGSSDTK

SVWIAtTGSD

FASQ

SNSD

SsStASR

NSSSSASR

Q(A

0A0A

3BP13)

3246

437317

KEsEG

VWGWsLQCL

LLLF

TKVNLR

scDQL

LICA

LVIRE

(Q5A

KS1)

299

465996

466136

1500

0plusmn

1100

3200plusmn

2700

000

014

KVILIG

NSM

TsRT

TsSV

RFFSIM

LTVS

LPItN

ILVsSEL

SKV

(A0A

0A3B

Z01)

3311



Biologicalfunction







(A0A0A6JC81)1 182




(A0A0A3BNV6)1 203

DNA binding

57544 57645 9700 plusmn1000



(C4YDL3)1 154

129759

12977518000 plusmn8800

51000 plusmn5400 000042


1

154




156



(A0A0A4CD62) 1 315

190171 190374 7800 plusmn3900


(C4YMX3) 2 319





4 Conclusions


Disclosure



Competing Interests


Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


ble1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

337660

337698

4300

0plusmn

1200

03200

0plusmn

2800

0023

KSSPT

SsAT

TTAT

tSVsISSLSLtMGKP

KNSK

LMediatorc

omplexlowast

(A0A

0A3C

P70)

3315

349815

350080

3400

0plusmn

8900

1800

0plusmn

1600

00054

KLG

SVLT

tRSQ

LIEY

ELtTRtIFIN

CSA

ALK

ISpt6

(D3IZV

2)3

311

350340

350474

4300

0plusmn

8300

3100

0plusmn

2500

0014

REV

IIKL

SITR

tHtPPP

DST

tTTT

PTTS

IEKT

Znfin

gerd

omainlowast

(A0A

0A6J6A

5)3

357

393914

394046

3600

0plusmn

4000

1700

0plusmn

8000

00026

VSSY

ILDGNNST

KLPsPV

LtHtTF

DSR

sDEG

QR

Basic

leucinez

ipperd

omainlowast

(A0A

0A3D

LJ5)

3399

411551

411695

5100

0plusmn

1200

02400

0plusmn

1700

00017

RAPQ

sIQLP

PIQsFtKsQ

AVFP

QSV

RDSA

PAANFN

RY

Transcrip

tionfactor

andDNA

bind


(A0A

0A6ITE

8)3

312

465996

465915

1500

0plusmn

1100

3200plusmn

2700

000

014

MKImmIPTH

HQtYNIN

THQPP

QQHQYL

PPPG

tSY

TSPR

A312

411551

411487

5100

0plusmn

1200

02400

0plusmn

1700

00017

KsStQ

MSSCtNsV

TQTL

DRL

PKIV

STQQNNL

TPTS

KI

Zn(2)-DNAbind

ingdo

mainlowast

(A0A

0A6K

Y81)

3303

465996

466019

1500

0plusmn

1100

3200plusmn

2700

000

014

RRS

VSYS

PGPsSIKS

QLP

HLT

SSST

TtssVQ

SPP

PPPP

SQPP

RG

361

417360

417373

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

KtV

TsIN

GSP

PPLE

tAPsSH

HNVPIDFIHFK

KESD

RT

Uncharacterized

protein

(A0A

0A3E

PD4)

3312

436859

436789

3300

0plusmn

2100

07100plusmn

1300

0020

KNMQFP

PYQVS

sHNsSEtSQ

sIPN

TPSITR

QVE

SNTR

S

Transcrip

tionalregulatory

proteinLE

U3

(A0A

0A3B

TY8)

3354

436790

KtPTT

TTTT

TTtANGNTS

NGNTS

NGNsTGKT

ATA

ATAT

KSNtK

Transcrip


(A0A

0A3C

3Y1)

319

Protein

synthesis

80477

80541

4200

0plusmn

2200

01900

0plusmn

3100

0034

KQTS

LNDKC

Manno

syltransfe

rase

familylowast

(A0A

0A3C

UB4

)1

254

80445

RVKI

DSD

KS

ProteintransporterS

EC24

(A0A

0A3E

HG4)

151

117588

117398

6400

0plusmn

3900

2200

0plusmn

1100lt000

001

KTK

QFN

DsK

KK

Vesic

letetheringprotein

familylowast

(A0A

0A6K

IG6)

1201

128483

128372

2700

0plusmn

9500

1400

0plusmn

2800

0016

KtAGsN

HNsEKK

Ribo

somalRN

A-processin

gprotein12

(A0A

0A4B

GJ7)

1251

412103

411993

3100

0plusmn

2300

01700

0plusmn

7200

013

KFF

sDNIANDLA

tTTT

TTTT

TNTG

AtSV

HPIL

QVDAIK

YCA

SCS


(A0A

0A6L

8P2)

3324

436859

437093

3300

0plusmn

2100

07100plusmn

1300

0020

KES

SSTA

DQPS

VVPP

QES

HKD

TVET

PKPE

VtEtsV

EAtKE

Transla


4G(A

0A0A

4CDK4

)3

299

436703

KNPT

PTPT

PTPT

PTPN

NLA

QGVDsSST

LDV

EtTL

tGLtRR

I


sphate

synthase

cycla

sesubu

nit

(A0A

0A6I238)

321


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Sign

allin

g

350340

350460

4300

0plusmn

8300

3100

0plusmn

2500

0013

KRS

SITtPtPP

LTTT

HSSNGNGNGNV

NVNVNsK

R

Peroxisometransmem

brane

receptorlowast

(A0A

0A3Z

WC2

)3

330

411551

411676

3100

0plusmn

2000

01700

0plusmn

7200

0017

KQST

TNTsTL

ssTtAAST

LATS

NNTQ

PDTY

TSTS

TSIRG

Pleckstrin

homologydo

mainlowast

(A0A

0A6L

5L9)

3333

441651

441487

2900

0plusmn

2700

01600

0plusmn

1100

010

RQHPD

PLSN

QsN

FNsN

TINNYS

NYR

SsTRS

GLD

PsQRH

RhoGTP

asea

ctivating

proteindo

mainlowast

(A0A

0A3B

MU5)

3412

465996

466032

1500

0plusmn

1100

3200plusmn

2700

000

014

KDLP

IGYILH

mIN

LcPN

IVsLNLG

NLSLsTD

YEISRS

TIHKY

F-bo

xproteinCO

S111

(A0A

0A3C

YY6)

3368

465821

KWNKE

KIEL

DsPLIVS

YVSSLC

NGGGGGIIT

NsTNST

ttNSK

Pentatric

opeptid

erepeat

(PPR

)protein

familylowast

(A0A

0A3C

GE7

)111

Misc

ellaneou

s

128483

128542

2700

0plusmn

9500

1400

0plusmn

2800

0016

RNDsD

tsLsK

EInsulin

indu

cedprotein

familylowast

(A0A

0A3C

RM0)

116

6RYtmNEV

FKV

GYF

domainlowast

(A0A

0A3C

0N8)

333128

333159

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

ETsQ

FMGNAES

EDLtGNGLL

TSTL

AVLS

SIS

Orfin

Calbicans

major

repeat

sequ

enceR

B2region

(Q5A

475)

3315

333128

333244

6300

0plusmn

1200

1400

0plusmn

1400

023

KLS

SLGNHGTtTT

SSLS

SSsSsSIsNNT

SIAKI

CBSdo

mainlowast

(A0A

0A6JTB

4)3

346

337660

337659

4300

0plusmn

1200

03300

0plusmn

2800

0RKQ

QDQNEV

AGAAAAT

TTTA

tAtAT

AAT

NWKP

KN

321

349815

349857

3400

0plusmn

8900

1800

0plusmn

1600

00054

KSSsLIK

NsTsSNQSsPA

TSTN

TSIV

DVPIEK


(A0A

0A3C

A48)

3321

417360

417295

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

RIN

NNNDKs

sILsNITTT

NTT

TGTG

TTNTtT

VPS

IKTK

R(A

0A0A

6IY5

3)424

349815

349948

3400

0plusmn

8900

1800

0plusmn

1600

00054

KAFS

SsKL

TSDSA

NStNStNsTSM

SILG

NDKD

CDKinhibitorP

HO81

(A0A

0A6M

Y31)

3316

389536

389486

3600

0plusmn

4100

2600

0plusmn

3100

00037

RISRP

NGVG

GISTS

GSSSP

TTEF

VTP

QAs

KsSV

DQNKK

RUbiqu


(C4Y

NW3)

3329

442103

442109

3200

0plusmn

1900

01700

0plusmn

7200

0080

RHLILG

YKItV

VtDHQSLTsVMTS

SSRP

ENNRM

IRW

Aspartic

peptidasefam

ilylowast

(A0A

0A4C

VF8

)3

383

393914

394016

3600

0plusmn

2400

01700

0plusmn

1800

000026

RNVNGSG

StNtN

TMtRLD

sTTIASSLF

CRQLY

FNLL

SKD

Globinfamilylowast

(A0A

0A4C

D75)

3311

437257

437390

9600plusmn

4600

5400plusmn

1800

0058

RNNsT

VSST

NSL

mSN

NsD

TNtAAT

AAT

AAT

SGS

TTNNVKR

M(A

0A0A

3XE4

6)338


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

441615

441482

2900

0plusmn

2700

01600

0plusmn

1100

010

RNGtYSSsStSSSsTSSVS

SSSA

TTANGES

LNST

THNIQ

LERT

Sec2plowast

(A0A

0A6JGN5)

3289

465996

466133

1500

0plusmn

1100

3200plusmn

2700

000

014

KQNYV

DPG

QIAIKGLK

GFV

NLG

AtCF

MssILQtLIH

NPL

IKY

Ubiqu

itincarboxyl-te

rminal

hydrolase

(C4Y

KS7)

3369

466093

KYD

KPmED

TEEIDDVtSISK

sINEQ

IDDPF

sQFN

SVTL

RY

Protein-serin

ethreon

ine

kinase

(A0A

0A4A

NB2

)35

1

Uncharacterized

117588

117373

6400

0plusmn

4000

2200

0plusmn

1100lt000

001

KtIm

tItIK

I(Q

59T3

9)1

207

333128

333260

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

RIsNIITL

NSSLS

SSsSsSSSsSSLLL

LTL

KS

(Q5A

AP9

)3

311

389536

389370

3600

0plusmn

4100

2600

0plusmn

3100

00034

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

314

393914

394074

3600

0plusmn

2400

01700

0plusmn

1800

000026

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

346

411551

411679

3100

0plusmn

2000

01700

0plusmn

7200

0017

KTV

CNWQtLGHTD

EFEE

stQFA

RGVS

DtAL

VGGITKL

(Q59K0

1)

3

218

411482

TCG

KSCF

INSttANKL

IsYN

LFQSStEVDS

IGPT

GSK

T(Q

59KZ

4)386

41162

KTT

PVMGNSsTP

STVTA

NTN

TGAY

STSsDt

AAKP

TKKA

TKR

(A0A

0A6JKT

0)391

411504

RILLL

LPLL

GVtILTS

KSSLES

ISLN

sPSD

SIALssSKS

(Q5A

H83)

234

441651

441518

2900

0plusmn

2700

01600

0plusmn

1100

010

KST

SSANIKtKPK

PNTA

TtAT

TPTA

tTAT

TtA

TTSSID

PTEK

D(A

0A0A

4CH02)

3259

442103

441999

3200

0plusmn

1900

01700

0plusmn

7200

0080

RLS

GtN

NPG

sGsG

sGGGGGGGANNNSLPG

YSV

GSSIG

RGRG

LGRG

(A0A

0A6LWB8

)3

189

436859

436693

3300

0plusmn

2100

07100plusmn

1300

0020

KNYN

QFK

LIEL

DDSM

NTT

TTTT

TTTT

tTttT

tTST

IGKF

(A0A

0A6M

D82)

3376

436787

KSK

sTTIHNQSN

IHSE

QISIN

DEN

NNKS

tstS

TSTD

TKK

(A0A

0A3E

QM3)

132

437257

437082

9600plusmn

4600

5400plusmn1800

0058

KSSGGSSDTK

SVWIAtTGSD

FASQ

SNSD

SsStASR

NSSSSASR

Q(A

0A0A

3BP13)

3246

437317

KEsEG

VWGWsLQCL

LLLF

TKVNLR

scDQL

LICA

LVIRE

(Q5A

KS1)

299

465996

466136

1500

0plusmn

1100

3200plusmn

2700

000

014

KVILIG

NSM

TsRT

TsSV

RFFSIM

LTVS

LPItN

ILVsSEL

SKV

(A0A

0A3B

Z01)

3311



Biologicalfunction







(A0A0A6JC81)1 182




(A0A0A3BNV6)1 203

DNA binding

57544 57645 9700 plusmn1000



(C4YDL3)1 154

129759

12977518000 plusmn8800

51000 plusmn5400 000042


1

154




156



(A0A0A4CD62) 1 315

190171 190374 7800 plusmn3900


(C4YMX3) 2 319





4 Conclusions


Disclosure



Competing Interests


Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

Sign

allin

g

350340

350460

4300

0plusmn

8300

3100

0plusmn

2500

0013

KRS

SITtPtPP

LTTT

HSSNGNGNGNV

NVNVNsK

R

Peroxisometransmem

brane

receptorlowast

(A0A

0A3Z

WC2

)3

330

411551

411676

3100

0plusmn

2000

01700

0plusmn

7200

0017

KQST

TNTsTL

ssTtAAST

LATS

NNTQ

PDTY

TSTS

TSIRG

Pleckstrin

homologydo

mainlowast

(A0A

0A6L

5L9)

3333

441651

441487

2900

0plusmn

2700

01600

0plusmn

1100

010

RQHPD

PLSN

QsN

FNsN

TINNYS

NYR

SsTRS

GLD

PsQRH

RhoGTP

asea

ctivating

proteindo

mainlowast

(A0A

0A3B

MU5)

3412

465996

466032

1500

0plusmn

1100

3200plusmn

2700

000

014

KDLP

IGYILH

mIN

LcPN

IVsLNLG

NLSLsTD

YEISRS

TIHKY

F-bo

xproteinCO

S111

(A0A

0A3C

YY6)

3368

465821

KWNKE

KIEL

DsPLIVS

YVSSLC

NGGGGGIIT

NsTNST

ttNSK

Pentatric

opeptid

erepeat

(PPR

)protein

familylowast

(A0A

0A3C

GE7

)111

Misc

ellaneou

s

128483

128542

2700

0plusmn

9500

1400

0plusmn

2800

0016

RNDsD

tsLsK

EInsulin

indu

cedprotein

familylowast

(A0A

0A3C

RM0)

116

6RYtmNEV

FKV

GYF

domainlowast

(A0A

0A3C

0N8)

333128

333159

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

ETsQ

FMGNAES

EDLtGNGLL

TSTL

AVLS

SIS

Orfin

Calbicans

major

repeat

sequ

enceR

B2region

(Q5A

475)

3315

333128

333244

6300

0plusmn

1200

1400

0plusmn

1400

023

KLS

SLGNHGTtTT

SSLS

SSsSsSIsNNT

SIAKI

CBSdo

mainlowast

(A0A

0A6JTB

4)3

346

337660

337659

4300

0plusmn

1200

03300

0plusmn

2800

0RKQ

QDQNEV

AGAAAAT

TTTA

tAtAT

AAT

NWKP

KN

321

349815

349857

3400

0plusmn

8900

1800

0plusmn

1600

00054

KSSsLIK

NsTsSNQSsPA

TSTN

TSIV

DVPIEK


(A0A

0A3C

A48)

3321

417360

417295

2400

0plusmn

1200

1300

0plusmn

1200

000

0014

RIN

NNNDKs

sILsNITTT

NTT

TGTG

TTNTtT

VPS

IKTK

R(A

0A0A

6IY5

3)424

349815

349948

3400

0plusmn

8900

1800

0plusmn

1600

00054

KAFS

SsKL

TSDSA

NStNStNsTSM

SILG

NDKD

CDKinhibitorP

HO81

(A0A

0A6M

Y31)

3316

389536

389486

3600

0plusmn

4100

2600

0plusmn

3100

00037

RISRP

NGVG

GISTS

GSSSP

TTEF

VTP

QAs

KsSV

DQNKK

RUbiqu


(C4Y

NW3)

3329

442103

442109

3200

0plusmn

1900

01700

0plusmn

7200

0080

RHLILG

YKItV

VtDHQSLTsVMTS

SSRP

ENNRM

IRW

Aspartic

peptidasefam

ilylowast

(A0A

0A4C

VF8

)3

383

393914

394016

3600

0plusmn

2400

01700

0plusmn

1800

000026

RNVNGSG

StNtN

TMtRLD

sTTIASSLF

CRQLY

FNLL

SKD

Globinfamilylowast

(A0A

0A4C

D75)

3311

437257

437390

9600plusmn

4600

5400plusmn

1800

0058

RNNsT

VSST

NSL

mSN

NsD

TNtAAT

AAT

AAT

SGS

TTNNVKR

M(A

0A0A

3XE4

6)338


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

441615

441482

2900

0plusmn

2700

01600

0plusmn

1100

010

RNGtYSSsStSSSsTSSVS

SSSA

TTANGES

LNST

THNIQ

LERT

Sec2plowast

(A0A

0A6JGN5)

3289

465996

466133

1500

0plusmn

1100

3200plusmn

2700

000

014

KQNYV

DPG

QIAIKGLK

GFV

NLG

AtCF

MssILQtLIH

NPL

IKY

Ubiqu

itincarboxyl-te

rminal

hydrolase

(C4Y

KS7)

3369

466093

KYD

KPmED

TEEIDDVtSISK

sINEQ

IDDPF

sQFN

SVTL

RY

Protein-serin

ethreon

ine

kinase

(A0A

0A4A

NB2

)35

1

Uncharacterized

117588

117373

6400

0plusmn

4000

2200

0plusmn

1100lt000

001

KtIm

tItIK

I(Q

59T3

9)1

207

333128

333260

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

RIsNIITL

NSSLS

SSsSsSSSsSSLLL

LTL

KS

(Q5A

AP9

)3

311

389536

389370

3600

0plusmn

4100

2600

0plusmn

3100

00034

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

314

393914

394074

3600

0plusmn

2400

01700

0plusmn

1800

000026

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

346

411551

411679

3100

0plusmn

2000

01700

0plusmn

7200

0017

KTV

CNWQtLGHTD

EFEE

stQFA

RGVS

DtAL

VGGITKL

(Q59K0

1)

3

218

411482

TCG

KSCF

INSttANKL

IsYN

LFQSStEVDS

IGPT

GSK

T(Q

59KZ

4)386

41162

KTT

PVMGNSsTP

STVTA

NTN

TGAY

STSsDt

AAKP

TKKA

TKR

(A0A

0A6JKT

0)391

411504

RILLL

LPLL

GVtILTS

KSSLES

ISLN

sPSD

SIALssSKS

(Q5A

H83)

234

441651

441518

2900

0plusmn

2700

01600

0plusmn

1100

010

KST

SSANIKtKPK

PNTA

TtAT

TPTA

tTAT

TtA

TTSSID

PTEK

D(A

0A0A

4CH02)

3259

442103

441999

3200

0plusmn

1900

01700

0plusmn

7200

0080

RLS

GtN

NPG

sGsG

sGGGGGGGANNNSLPG

YSV

GSSIG

RGRG

LGRG

(A0A

0A6LWB8

)3

189

436859

436693

3300

0plusmn

2100

07100plusmn

1300

0020

KNYN

QFK

LIEL

DDSM

NTT

TTTT

TTTT

tTttT

tTST

IGKF

(A0A

0A6M

D82)

3376

436787

KSK

sTTIHNQSN

IHSE

QISIN

DEN

NNKS

tstS

TSTD

TKK

(A0A

0A3E

QM3)

132

437257

437082

9600plusmn

4600

5400plusmn1800

0058

KSSGGSSDTK

SVWIAtTGSD

FASQ

SNSD

SsStASR

NSSSSASR

Q(A

0A0A

3BP13)

3246

437317

KEsEG

VWGWsLQCL

LLLF

TKVNLR

scDQL

LICA

LVIRE

(Q5A

KS1)

299

465996

466136

1500

0plusmn

1100

3200plusmn

2700

000

014

KVILIG

NSM

TsRT

TsSV

RFFSIM

LTVS

LPItN

ILVsSEL

SKV

(A0A

0A3B

Z01)

3311



Biologicalfunction







(A0A0A6JC81)1 182




(A0A0A3BNV6)1 203

DNA binding

57544 57645 9700 plusmn1000



(C4YDL3)1 154

129759

12977518000 plusmn8800

51000 plusmn5400 000042


1

154




156



(A0A0A4CD62) 1 315

190171 190374 7800 plusmn3900


(C4YMX3) 2 319





4 Conclusions


Disclosure



Competing Interests


Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Con

tinued

Biological

functio

n

Observed

precursor

mz

Theoretic

alprecursor

mz

MSsig

nal

intensities

119901values

Aminoacid

sequ

ence

Proteinname

(UniProt

AccessionNum

ber)

Charges119883

Corr

Con

trol

HTN

5tre

ated

441615

441482

2900

0plusmn

2700

01600

0plusmn

1100

010

RNGtYSSsStSSSsTSSVS

SSSA

TTANGES

LNST

THNIQ

LERT

Sec2plowast

(A0A

0A6JGN5)

3289

465996

466133

1500

0plusmn

1100

3200plusmn

2700

000

014

KQNYV

DPG

QIAIKGLK

GFV

NLG

AtCF

MssILQtLIH

NPL

IKY

Ubiqu

itincarboxyl-te

rminal

hydrolase

(C4Y

KS7)

3369

466093

KYD

KPmED

TEEIDDVtSISK

sINEQ

IDDPF

sQFN

SVTL

RY

Protein-serin

ethreon

ine

kinase

(A0A

0A4A

NB2

)35

1

Uncharacterized

117588

117373

6400

0plusmn

4000

2200

0plusmn

1100lt000

001

KtIm

tItIK

I(Q

59T3

9)1

207

333128

333260

6300

0plusmn

1200

1400

0plusmn

1400lt000

001

RIsNIITL

NSSLS

SSsSsSSSsSSLLL

LTL

KS

(Q5A

AP9

)3

311

389536

389370

3600

0plusmn

4100

2600

0plusmn

3100

00034

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

314

393914

394074

3600

0plusmn

2400

01700

0plusmn

1800

000026

KTT

PVMGNSStPST

VtANtN

tGAY

STSSDTA

AKP

TKKA

(A0A

0A6JKT

0)3

346

411551

411679

3100

0plusmn

2000

01700

0plusmn

7200

0017

KTV

CNWQtLGHTD

EFEE

stQFA

RGVS

DtAL

VGGITKL

(Q59K0

1)

3

218

411482

TCG

KSCF

INSttANKL

IsYN

LFQSStEVDS

IGPT

GSK

T(Q

59KZ

4)386

41162

KTT

PVMGNSsTP

STVTA

NTN

TGAY

STSsDt

AAKP

TKKA

TKR

(A0A

0A6JKT

0)391

411504

RILLL

LPLL

GVtILTS

KSSLES

ISLN

sPSD

SIALssSKS

(Q5A

H83)

234

441651

441518

2900

0plusmn

2700

01600

0plusmn

1100

010

KST

SSANIKtKPK

PNTA

TtAT

TPTA

tTAT

TtA

TTSSID

PTEK

D(A

0A0A

4CH02)

3259

442103

441999

3200

0plusmn

1900

01700

0plusmn

7200

0080

RLS

GtN

NPG

sGsG

sGGGGGGGANNNSLPG

YSV

GSSIG

RGRG

LGRG

(A0A

0A6LWB8

)3

189

436859

436693

3300

0plusmn

2100

07100plusmn

1300

0020

KNYN

QFK

LIEL

DDSM

NTT

TTTT

TTTT

tTttT

tTST

IGKF

(A0A

0A6M

D82)

3376

436787

KSK

sTTIHNQSN

IHSE

QISIN

DEN

NNKS

tstS

TSTD

TKK

(A0A

0A3E

QM3)

132

437257

437082

9600plusmn

4600

5400plusmn1800

0058

KSSGGSSDTK

SVWIAtTGSD

FASQ

SNSD

SsStASR

NSSSSASR

Q(A

0A0A

3BP13)

3246

437317

KEsEG

VWGWsLQCL

LLLF

TKVNLR

scDQL

LICA

LVIRE

(Q5A

KS1)

299

465996

466136

1500

0plusmn

1100

3200plusmn

2700

000

014

KVILIG

NSM

TsRT

TsSV

RFFSIM

LTVS

LPItN

ILVsSEL

SKV

(A0A

0A3B

Z01)

3311



Biologicalfunction







(A0A0A6JC81)1 182




(A0A0A3BNV6)1 203

DNA binding

57544 57645 9700 plusmn1000



(C4YDL3)1 154

129759

12977518000 plusmn8800

51000 plusmn5400 000042


1

154




156



(A0A0A4CD62) 1 315

190171 190374 7800 plusmn3900


(C4YMX3) 2 319





4 Conclusions


Disclosure



Competing Interests


Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Biologicalfunction







(A0A0A6JC81)1 182




(A0A0A3BNV6)1 203

DNA binding

57544 57645 9700 plusmn1000



(C4YDL3)1 154

129759

12977518000 plusmn8800

51000 plusmn5400 000042


1

154




156



(A0A0A4CD62) 1 315

190171 190374 7800 plusmn3900


(C4YMX3) 2 319





4 Conclusions


Disclosure



Competing Interests


Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Competing Interests


Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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