Chapter 16
Proteomic Strategies to Characterize Signaling Pathways
H.C. Harsha, Sneha M. Pinto, and Akhilesh Pandey
Abstract
Cells respond to external stimuli by transducing signals through a series of intracellular molecules andeliciting an appropriate response. The cascade of events through which the signals are transduced includepost-translational modifications such as phosphorylation and ubiquitylation in addition to formation ofmulti-protein complexes. Improvements in biological mass spectrometry and protein/peptide microarraytechnology have tremendously improved our ability to probe proteins, protein complexes, and signalingpathways in a high-throughput fashion. Today, a single mass spectrometry-based investigation of a signalingpathway has the potential to uncover the large majority of known signaling intermediates painstakinglycharacterized over decades in addition to discovering a number of novel ones. Here, we discuss variousproteomic strategies to characterize signaling pathways and provide protocols for phosphoproteomicanalysis.
Key words Signaling pathways, Mass spectrometry, Phosphorylation, Enrichment of phosphopro-teomes, Quantitative phosphoproteomics, Cancer
1 Introduction
1.1 Mass
Spectrometry
to Characterize
Signaling Pathway
Intermediates
The last decade has witnessed tremendous progress in massspectrometry approaches that has enabled unbiased global proteinprofiling studies. Enrichment and fractionation strategies have alsoevolved that enable deeper sampling of specific sub-proteomes ofinterest. Phosphorylation is one of the major post-translationalmodifications (PTMs) through which signals are transduced in acell. Methods to enrich phosphorylated peptides have contributedsignificantly to our ability to characterize phosphoproteomes ofcells and tissues. One major methodology that is used for enrich-ment is the use of antibodies that specifically enrich for tyrosine[1–3] or serine/threonine [4] phosphorylated proteins. These anti-phosphotyrosine and anti-phospho-serine/threonine antibodiesalso enable visualization of global activation of signaling moleculesupon a stimulus. Following receptor stimulation using an appro-priate ligand, signaling intermediates that are phosphorylated in aninducible manner can be immunoprecipitated and visualized by
Rune Matthiesen (ed.), Mass Spectrometry Data Analysis in Proteomics, Methods in Molecular Biology, vol. 1007,DOI 10.1007/978-1-62703-392-3_16, # Springer Science+Business Media, LLC 2013
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Western blotting using the same antibodies. Although thesephosphorylated proteins can be visualized as distinct bands byWestern blotting, the identity of these proteins cannot be deter-mined. Tyrosine kinase signaling pathways were some of the firstpathways to be characterized using mass spectrometry. This waslargely enabled owing to the availability of robust antibodies toenrich tyrosine phosphoproteome. Since then, phosphoproteomicapproaches have been extensively used to characterize severalsignaling intermediates of growth factor and cytokine receptorsignaling pathways [4–6]. In addition, this method has been usedto characterize various immune signaling pathways as well as cancersignaling pathways. Salomon et al. employed phosphoproteomicsstrategy and showed tyrosine phosphorylation dynamics of tyrosinephosphosites in human T-cell signaling and BCR-ABL signaling[7]. In recent years, phosphoproteomic approaches in conjunctionwith stable isotope labeling strategies have resulted in characteriza-tion of thousands of molecules in a signaling pathway in a singleexperiment [8, 9].
1.2 Experimental
Strategies for
Enrichment of
Phosphoproteomes
Low stoichiometry of phosphoproteome necessitates use of enrich-ment methods to obtain detectable amounts of phosphopeptidesfrom biological samples. Effective enrichment of tyrosine phospho-proteins/peptides has been achieved using anti-phosphotyrosineantibodies. In particular, three monoclonal antibodies, 4G10,p-Tyr-100, and pY20, have been extensively used for tyrosine phos-phoprotein/peptide enrichment. These antibodies are known tohave overlapping but somewhat different specificities based on thesequence context surrounding the phosphotyrosine residues [10].A combination of these antibodies is likely to yield better coverageof tyrosine phosphoproteome than using each antibody in isolation.
In addition to antibody-based approaches, strategies that relyon chromatography-based methods as well as metal affinity-basedenrichment approaches referred to as immobilized metal affinitychromatography (IMAC) have also evolved for enrichment ofphosphopeptides [11–14]. Chromatography-based methods forpreferential enrichment of phosphopeptides include strong cationexchange (SCX) chromatography [15], strong anion exchange(SAX) chromatography [16], hydrophilic interaction liquid chro-matography (HILIC) [17], and electrostatic repulsion-hydrophilicinteraction chromatography (ERLIC) [18]. Chromatography-based methods often utilize the difference in the physicochemicalcharacteristics of the phosphopeptide as discriminating factorsto separate phosphopeptides from non-phosphorylated peptides.For example, phosphopeptides elute in the early fractions in SCXchromatography (at pH 2.7)-based fractionation as tryptic phos-phopeptides possess a lower net charge owing to negatively chargedphosphate moieties.
360 H.C. Harsha et al.
Several immobilized metal affinity matrices have beenemployed including Fe3+, Ga3+, and Zr4+ [13, 19, 20]. One ofthe commonly employed methods for phosphopeptides is TiO2-based enrichment. This method is now routinely used to profilethousands of phosphorylation sites in various experimental con-texts. Some studies have also employed combinatorial strategieswhere SCX chromatography is first used to enrich phosphopeptidesfollowed by subsequent IMAC/TiO2-based enrichment for betterselectivity and specificity [21–23].
Antibody-based strategies are superior for enrichment of tyro-sine phosphopeptides, whereas TiO2- [24–26] and IMAC-basedstrategies have been found to be efficient at enriching serine/thre-onine phosphopeptides. Large-scale signaling studies have takenadvantage of this ability to identify and characterize large numberof mediators of cell signaling. In addition to phosphorylation, massspectrometric methods are also being used to investigate role ofacetylation, glycosylation, and ubiquitination in signal transductionpathways. For instance, the interplay of phosphorylation andO-GlcNAcylation and its implications on signaling is already welldocumented [27].
1.3 Quantitative
Phosphoproteomic
Studies that Have
Characterized Specific
Signaling Pathways
Several signaling pathways have now been characterized using massspectrometry-based global phosphoproteomic profiling. Thesestudies typically use cells that are either stimulated by specificligands followed by phosphoproteomics to identify pathway sub-strates (Fig. 1). Proteins that undergo phosphorylation upon acti-vation of specific signaling pathways are concluded as downstreameffectors of respective signaling pathways. EGF signaling was oneof the first signaling pathways to be characterized in a comprehen-sive fashion by mass spectrometry. Blagoev et al. employed aSILAC (stable isotope labeling by amino acids in cell culture)-based strategy coupled to an antibody-based affinity purificationmethod for enriching EGF-induced phosphoproteins. The experi-ment assessed protein phosphorylation dynamics across five timepoints post-EGF stimulation and identified about 81 effectors in asingle experiment [28]. Subsequently, Zhang et al. studied EGFRactivation in a time-dependent manner by employing both anti-body- and IMAC-based enrichment strategies and identified sev-eral downstream effectors [29]. Olsen et al. characterizedhundreds of site-specific changes downstream of EGF stimulationby employing a TiO2-based phosphopeptide enrichment strategy[30]. More recently, similar phosphoproteomic strategies havebeen employed to characterize kinase signaling pathways includingc-Src [31–35], ERBB2 [36, 37], Insulin [38], EphB2 [39, 40],FGF [41, 42], FGF-2 [43, 44], IL-2 [45], TGF-β [46], and TSLP[47] among others.
Proteomic Strategies to Characterize Signaling Pathways 361
No treatment Treatment with ligand
Grow in regular media Cells grown in stable isotope labeledamino acid contaning media
Rel
ativ
e A
bund
ance
m/z0
100 Pathway substrate
Unchanged by ligand
LC-MS analysis
Phosphopeptide enrichment
Fig. 1 Identification of pathway substrates by phosphoproteomics. Phosphopep-tides are enriched from stimulated and unstimulated cellular states and analyzedby mass spectrometry. Proteins/peptides from ligand-stimulated state areencoded using stable isotopes (asterisk) which introduces a mass shift thusfacilitating determination of relative abundance by mass spectrometry. Redpeptides represent an example of a pathway substrate that undergoes anincrease in phosphorylation after stimulation of cells with a specific ligand
1.4 Mass
Spectrometry to
Determine Dynamic
States of Proteins
in Signaling Pathways
Incorporation of quantitative proteomic strategies such as SILAC,iTRAQ, and TMT has tremendously improved our ability to deter-mine quantitative differences in protein expression across cellularstates [48–50]. These labeling strategies and label-free strategieshave played a vital role in subsequent years to characterize dynamicchanges in protein modifications as well as variations in proteincomplexes in signaling pathways [28, 29, 51, 52]. To characterizedynamic states of protein complexes in signaling pathways, recruit-ment of proteins upon activation of specific pathways can be deter-mined by mass spectrometry.
1.5 Determining
Kinase-Substrate
Motifs from High-
Throughput
Phosphoproteomic
Data
Kinases are known to recognize their substrates based on specificsequence motifs flanking the phosphorylation site. Identification ofspecific kinase substrate motifs provides opportunity to predictpotential targets of kinases. Global phosphoproteomic studies gen-erate large amounts of phosphopeptide data that can be utilized toidentify enriched sequences flanking phosphorylation sites. Tilldate, more than 300 kinase substrate motifs have been describedin the literature [53].
Over the past decade, several computational methods/toolshave been developed to predict potential PTM sites in proteinsequences. These predict phosphorylation sites in a kinase-dependent or kinase-independent fashion [54]. Kinase-dependentapproaches are based on the presumption that each kinase has aunique sequence specificity and thus substrates of these kinases canbe predicted using information available on kinase-specific motifsfrom literature curated databases such as Phospho.ELM [55],PhosphoSite [56], and HPRD [57]. Several prediction tools havebeen developed such as Scansite [58] and NetPhorest [59] whichpredict kinases from linear motifs using artificial neural networksor position-specific scoring matrices (PSSM). NetPhos [60] andKinasePhos [61] are other prediction tools that predict kinasephosphorylation sites based on available dataset of known phos-phorylation sites.
1.6 Mass
Spectrometry
Approaches to
Determine Aberrantly
Activated Signaling
Pathways in Diseases
Global phosphoproteomic profiling studies are now beingextensively carried out to characterize aberrantly regulated signaltransduction pathways in various diseases including cancers. Dys-regulation of kinase signaling pathways in cancers is now wellunderstood [62] and has led to the development of targetedtherapeutic strategies where a mutant kinase or an aberrantly acti-vated kinase is targeted for therapeutic purposes. Examples of thisinclude chronic myelogenous leukemia (CML) where BCR-ABL, afusion kinase is the therapeutic target and is treated with a smallmolecule inhibitor imatinib [63]; non-small cell lung cancer wherea mutant EGFR is targeted using small molecule inhibitor erlotinib[64]; and breast cancers where HER2/neu is targeted using anantibody [65].
Proteomic Strategies to Characterize Signaling Pathways 363
These successful outcomes have provided an impetus todeveloping strategies for identifying aberrant kinases in other diseasesincluding various cancers. We have previously shown the utility ofphosphoproteomics in identifying aberrantly activated kinase signal-ing pathways in pancreatic cancer [66]. Using an antibody-basedstrategy to enrich and compare tyrosine phosphoproteins from anon-neoplastic cell line and a neoplastic cell line of pancreas, aberrantactivation of EGFR pathway was identified in a subset of pancreaticcancers. The role of this activation of EGFR in driving cancer cellproliferation was tested by carrying out mouse xenograft studieswhere the implanted tumors were shown to regress upon treatmentwith an EGFR inhibitor. Other cancers where phosphoproteomicprofiling studies have been employed to identify driver kinases orkinase-driven signaling pathways include leukemia [67], sarcomas[68], non-small cell lung cancers [69], renal cell carcinoma [70],glioblastoma [71], melanoma [72], and colon cancer [73].
Phosphoproteomic approaches have also been employed tostudy phosphorylation changes resulting from specific drug treat-ment [74]. Huang et al. employed phosphotyrosine strategy toexplore therapeutic strategy for glioblastoma [75]. Phosphopro-teomics is also being employed to investigate drug resistance andescape mechanisms in cancers. Rexer et al. generated six lapatinibresistant HER2 overexpressing human breast cancer cell lines toinvestigate resistance mechanisms. The resistant lines were found toutilize Src family kinase-driven pathways as escape mechanism [76].Andersen et al. carried out pathway-based phosphoprofiling toidentify biomarkers to predict responsiveness to targeted therapy.They showed pPRAS40 (Thr246) as a predictor of PI3K pathwayactivation and AKT inhibitor sensitivity [77].
1.7 Protein and
Peptide Microarray
Strategies to Study
Signaling
In addition to mass spectrometry, protein and peptide microarraystrategies also offer an opportunity to characterize signaling path-ways in a high-throughput manner. Protein microarrays have beenused in the past to characterize signaling pathways, to identifykinase substrates, and to study recruitment potential of intracellularphosphorylated regions of receptor tyrosine kinases [78–80].Reverse-phase protein microarrays (RPPAs) have been extensivelyused for studying aberrantly activated signaling pathways in cancers[81]. RPPAs are often printed with normal and cancer cell/tissuelysates and are probed using antibodies against activated forms orphosphorylated forms of specific proteins. Using this methodology,hundreds of cell/tissue lysates can be probed in a single experimentin an array format [82]. Protein microarrays have been used exten-sively for characterizing signaling pathways in various cancersincluding prostate cancer [83], ovarian cancer [84], and T-cellacute lymphoblastic leukemia [85]. In addition, protein microarraytechnology has also been used to identify molecular networksassociated with antiestrogen resistance in breast cancers [86], to
364 H.C. Harsha et al.
monitor response to kinase inhibitors [87], and to study aberrantactivation of signaling molecules in head and neck cancers [88].Peptide microarrays are also being used for high-throughputscreening of kinase substrates and also to characterize motifs recog-nized by specific kinases [33, 89].
1.8 Challenges
in Phosphorylation
Analysis
The most commonly used fragmentation method in proteomics iscollision-induced dissociation (CID). Fragmentation of serine/threonine phosphopeptides by CID generally yields fragmentsthat have lost the phosphate moieties. These precursor ions thathave lost the phosphate moiety generally dominate the MS/MSspectrum. This insufficient fragmentation of the phosphopeptidedirectly impacts peptide identification and phosphorylation sitedetermination. Electron transfer dissociation (ETD) has beenshown to be a better strategy for fragmentation while dealingwith labile modifications like O-GlcNAc and serine/threoninephosphorylation [26]. Trypsin remains the most widely used pro-tease in mass spectrometry-based proteomic analysis due to itsnumerous advantages; however, the routine use of trypsin forprotein digestion does not yield a comprehensive picture of allpotential phosphorylation events because some of the tryptic pep-tides are undetectable in MS (e.g., either too short or too long).Employing multiple proteases that have site-specific cleavage activ-ity have been shown to significantly improve sequence coverage inglobal proteomic analysis and the same strategy can be used tocomplement and improve phosphoproteome coverage [91].
Search engines such as Sequest, Mascot, and X!Tandem areemployed for protein identifications from MS fragment spectra.However, the fragmentation information may not be sufficient toaccurately assign the position of phosphorylation site. Sometimes,manual inspection MS/MS spectra is carried out to deduce themodified residue, although this is a daunting task and not feasiblefor large-scale phosphoproteomic experiments involving thousandsof MS/MS spectra. The exact localization of phosphorylation sitesnecessitates comparison of the observed spectra with theoreticalspectra containing all possible permutations of the phosphategroup being placed on possible sequence position. Computationaltools such as Ascore [92], MaxQuant [93], PhosCalc [94], andPhosphoRS [95] among others, have been developed which utilizealgorithms to improve peptide identification and localization of thesite of phosphorylation when multiple sites of phosphorylation maybe present. Ascore and PhosphoRS are probability-based scoringsystems which utilize peptide sequence identified in protein data-base search to generate a list of all possible combination of phos-phopeptides. The site localization by all these tools depends uponsite-determining fragment ions. Based on these ions, these algo-rithms calculate site-specific scores based on binomial probabilitiesand individual site probabilities.
Proteomic Strategies to Characterize Signaling Pathways 365
1.9 Signal
Transduction Pathway
Resources
Signaling pathways have been painstakingly characterized over theyears by careful work on individual effectors in a particular signalingpathway. This information is scattered in the scientific literature.There are various public resources including KEGG [96], Biocarta,and NetPath that provide organized information on signaling path-ways and pathway intermediates. NetPath is one of the mostdetailed pathway resources of human signaling pathways [97].
Phosphoproteomic studies generate enormous amount ofdata. One of the goals of phosphoproteomic studies in diseasecontexts is to identify pathways that are altered in pathologicalconditions. The readout obtained in these studies is the sum totalof signaling activity in those biological samples. Several bioinfor-matics approaches are being employed to determine activated sig-naling pathways. Most of these approaches rely on datasets fromknown literature and some form of enrichment analysis to identify ahigher likelihood of a given pathway to be altered. Various types ofexperimental data are utilized for this including co-expression,protein–protein interactions, colocalization and also known infor-mation on the involvement of molecules in specific pathways basedon the existing literature. We anticipate that, in the future, signaltransduction pathway resources will become truly comprehensiveand enable development of better tools and algorithms to discoveraberrant signaling pathways from high-throughput phosphopro-teomic and other proteomic studies.
2 Materials
2.1 Materials
for Characterizing
Signaling Pathways
Using Quantitative
Phosphoproteomics
1. Cell culture medium depleted of lysine and arginine aminoacids.
2. Stable isotope-labeled amino acids—13C6-lysine (CambridgeIsotopes, Catalog # CLM-2247-H-1) and 13C6-arginine(Cambridge Isotopes, Catalog # CLM-2265-H-1).
3. Fetal bovine serum.
4. Appropriate ligand to stimulate the pathway under investiga-tion.
5. Urea (Thermo Scientific, Catalog # 29700).
6. HEPES (Sigma-Aldrich, Catalog # H4034-100G).
7. Complete protease inhibitor cocktail tablets (Roche, Catalog #12715300).
8. Sodium orthovanadate (Sigma-Aldrich, Catalog # S6508).
9. Sodium pyrophosphate (Sigma-Aldrich, Catalog # S6422).
10. β-glycerophosphate (Sigma-Aldrich, Catalog #50020).
11. Dithiothreitol (DTT) (Sigma-Aldrich, Catalog # D9779-10G).
366 H.C. Harsha et al.
12. Iodoacetamide (IAA) (Sigma-Aldrich, Catalog # I1149-5G).
13. Sequencing grademodified trypsin (Promega,Catalog#V5111).
14. Sep-Pak Classic C18 cartridges (WATERS, Catalog #WAT051910).
15. Trifluoroacetic acid (TFA) (Fisher Scientific Catalog #A116-50).
16. Pierce BCA protein assay kit (Thermo Scientific, Catalog #23225).
17. Triethylammonium bicarbonate (TEABC stock) buffer 1.0 M,pH 8.4–8.6 (Sigma-Aldrich, Catalog #. 17902) 100 ml(>98.5 % purity).
18. XBridge C18, 5 μm 250 � 4.6 mm analytical column(Waters, Milford, MA, Catalog #. 186003117) for bRPLCfractionation.
19. Titansphere 10 or 5 μm (GL Sciences, Inc., Japan Catalog #5020-75010).
20. Anti-phosphotyrosine antibody 4G10 agarose conjugate(Millipore, Catalog # 16-101) or anti-phosphotyrosine mousemAb (p-Tyr-100) (sepharose conjugate) (Cell Signaling Tech-nology, Catalog # 9419).
21. 2, 5-Dihydroxybenzoic acid (DHB) (Sigma-Aldrich, Catalog#. 149357-100G).
22. Ammonium hydroxide (J.T. Baker, Catalog #. 9721-01).
23. Empore C18 Disk (Catalog # 98060402181).
2.2 Solvents 1. Solvent A: 7 mM TEABC in water (Add 7 ml TEABC stockto 1 L) (pH 9).
2. Solvent B: 7 mM TEABC in 90 % acetonitrile (ACN) (JTBaker, 99.9 % pure), (Add 7 ml TEABC stock to 1 L of 90 %ACN).
3. Wash solution 1(80 % ACN, 1 % TFA, HPLC grade).
4. DHB solution (5 % 2,5-dihydroxybenzoic acid (DHB), i.e.,50 mg of DHB in 1 ml of Wash solution 1).
5. Ammonia solution (2 % NH4OH, pH ~10.5, i.e., 20 μl ofNH4OH in 1 ml of 40 % ACN).
2.3 Equipment 1. Centrifuge with refrigeration.
2. Sonicator—Branson Sonifier 250.
3. Lyophilizer.
4. High-performance liquid chromatography system.
5. High-resolution mass spectrometer interfaced with nanoscaleliquid chromatography system.
Proteomic Strategies to Characterize Signaling Pathways 367
3 Methods
3.1 Stable Isotope
Labeling by Amino
Acids in Cell Culture
For SILAC-based experiments, it is essential to adapt the cells inmedia supplemented with heavy isotopic forms of lysine and argi-nine amino acids (http://www.silac.org). Also, refer to Harshaet al. [90] for a detailed protocol on adapting the cells to SILACmedia. To carry out deep proteomic profiling of tyrosine andserine/threonine phosphoproteome, it is desirable to obtain25–30 mg of proteins after combining lysates from both unlabeledand labeled cell population. Estimating one million cells to yieldapproximately 100 μg of protein, one would require ~1.5 � 108
for an experiment. Some laboratories are able to use significantlylower amount of starting material without compromising the depthof sampling—we anticipate that more and more laboratories will beable to do this in the future.
1. Culture 150 million cells in SILAC medium and same amountof cells in standard medium.
2. Once cells have reached the desired density, serum starve bothcell populations overnight.
3. Stimulate the labeled cell population with appropriate ligand forpredetermined time period (seeNote 1).
4. Lyse the cells in 9 M urea lysis buffer.
5. Sonicate cell lysate for efficient lysis (3 Woutput, duty cycle-40,3 cycles of 10–15 bursts per cycle).
6. Centrifuge the lysates at 12,000 � g for 15 min.
7. Collect the supernatant and estimate protein concentrationusing the BCA method.
8. Pool lysates equivalent to 13 mg protein from both unstimu-lated and stimulated conditions.
9. Reduce the proteins by adding DTT to a final concentration of5 mM and incubating at 56 �C for 45 min.
10. Bring the lysate to room temperature and alkylate the proteinsby adding IAA to a final concentration of 10 mM and incubat-ing in the dark for 30 min.
11. Dilute the lysate to obtain a final urea concentration of �2 Musing HEPES buffer.
12. Add trypsin to obtain an enzyme to substrate ratio of 1:20(w/w).
13. Incubate at 37 �C overnight.
14. Acidify the peptide digest by adding TFA to a final concentra-tion of 1 %.
15. Centrifuge at 4,000 � g for 15 min.
368 H.C. Harsha et al.
16. Collect the supernatant and clean up the sample using C18
Sep-Pak cartridge.
17. Aliquot 5 mg equivalent peptide digest into a separate tube.
18. Freeze the tubes containing 5 mg equivalent peptide and20 mg equivalent peptide digest using liquid nitrogen/dryice/�80 �C freezer.
19. Lyophilize the samples for 2–3 days (see Note 2).
3.2 Enrichment
of Tyrosine
Phosphopeptides
1. Dissolve 20 mg Sep-Pak purified lyophilized peptide digest in1.5 ml of HEPES buffer.
2. Sonicate the tube in a water bath sonicator for 10 min tofacilitate dissolution of peptides.
3. Centrifuge the tube at 12,000 � g for 5 min and recover thesupernatant into a fresh tube.
4. Check the pH using pH paper strip and ensure that the pH is7–8. If not, adjust the pH using 1 M Tris base (see Note 3).
5. Resuspend one vial of agarose conjugated pY100 anti-phosphotyrosine antibody from Cell Signaling Technologyor 50 μg equivalent agarose conjugated 4G10 anti-phosphotyrosine antibody fromMillipore in 1�HEPES buffer(see Note 4).
6. Gently mix by inverting the tubes 4–5 times to obtain homog-enous suspension (see Note 5).
7. Spin the tubes at 4,000 � g for 1 min at 4 �C and carefullyremove and discard the supernatant leaving 50 μl of bufferbehind (see Note 6).
8. Transfer the peptide sample into the microfuge tube containingthe washed agarose-conjugated anti-phosphotyrosine antibody.
9. Incubate the tube on a rotator at 4 �C for 2 h. Ensure that thespeed is adjusted to maintain a homogenous suspension ofthe beads.
10. Centrifuge the tube at 500 � g for 1 min and discard thesupernatant.
11. Wash the beads by adding 1.5 ml of HEPES buffer and mixingit gently by inverting the tube 4–5 times to obtain a homoge-nous suspension.
12. Aspirate the supernatant using a pipette.
13. Repeat steps 10–12 two times.
14. Wash the beads twice in 1 ml distilled water as described insteps 10–12.
15. For eluting the phosphopeptides, add 75 μl of 0.1 % TFA andtap it gently to obtain homogenous suspension.
Proteomic Strategies to Characterize Signaling Pathways 369
16. Leave the tube at room temperature for 5 min.
17. Centrifuge at 500 � g for 1 min.
18. Aspirate the supernatant without disturbing the beads using apipette with a gel loading tip.
19. Repeat steps 15–18.
20. Clean the eluate using C18 ZipTip.
3.3 C18 StageTip
Clean Up
1. Prepare a C18 StageTip using Empore C18 disk. Wash the C18
disk in the following order:
l 80 % ACN, 0.5 % acetic acid solution.
l 50 % ACN, 0.1 % formic acid.
l 0.15 % TFA.
2. Load the combined eluate onto a C18 StageTip. Elute thesample into microfuge tube and reload the eluate.
3. Wash the bound peptides with 35 μl of 0.15 % TFA. Check forthe presence of beads. Ensure that no beads are present.
4. Elute the bound peptides with 10 μl of 50 % ACN, 0.1 % formicacid (see Note 7).
3.4 Enrichment of
Phosphopeptides
using TiO2
1. Dissolve the Sep-Pak purified and lyophilized peptidedigest (5–10 mg) in 2 ml of basic RPLC Solution A (pH 9)(see Note 8).
2. Centrifuge the tube at 16,000 � g for 5 min at 4 �C andtransfer the supernatant to a microfuge tube.
3. Fractionate the peptide digest using basic reverse phase chro-matography. Collect fraction in a 96-well plate (1 ml capacity)(see Note 9).
4. Dry the fractions at freezing temperature.
5. Add 400 μl of 80 % acetonitrile with 0.1 % TFA to A1 to A12wells in the 96-well plate and concatenate fractions fromA1–H1, A2–H2. . .A12–H12 to obtain a total of 12 fractions.Dry the samples using speed vac fitted with refrigeratedvapor trap.
6. Reconstitute the fractions with 100 μl of 5 % DHB solution.Ensure the peptides are completely dissolved (see Note 10).
7. Suspend TiO2 beads (50 mg) in 100 μl of 5 % DHB solutionand incubate for 15 min on the rotator at room temp (seeNote 11).
8. Centrifuge briefly and discard supernatant.
9. Add 2 μl of beads to each tube (each fraction may have~500 μg–1 mg of peptides) (TiO2: peptide ratio of 1:1) andincubate the peptide–DHB–TiO2 mix on a rotator for 15 minat room temperature (see Note 12).
370 H.C. Harsha et al.
10. Centrifuge the tubes at 1,500 � g for 1 min.
11. Transfer the entire content to StageTip with C8 plug.
12. Elute the unbound peptides into an Eppendorf tube.
13. Wash the peptide–TiO2 beads with 100 μl of wash solution 1.
14. Repeat step 12.
15. Elute phosphopeptides from TiO2 beads in 30 μl of 2 %NH4OH solution into collection tubes containing 20 μl of0.1 % TFA.
16. Repeat the elution step twice.
17. Dry the samples in speedvac without heating.
18. Resuspend dried peptides in 0.1 % TFA and desalt them usingC18 StageTip (or ZipTip).
4 Notes
1. The cells under study can also be reverse labeled to carry out abiological replicate experiment.
2. Extended period of lyophilization facilitates removal of tracesof TFA as low pH affects immunoaffinity purification.
3. While adjusting pH of the peptide digest prior to immunoaffi-nity purification, Tris–base must be added in small aliquots of5 μl until the desired pH of 7–8 is reached.
4. Anti-phosphotyrosine antibodies pY100 and 4G10 have over-lapping and slightly varied specificities. Using a mixture of thesetwo antibodies yields larger number of tyrosine phosphopep-tides as compared to using only one of them.
5. Antibody conjugated beads should be mixed gently to create ahomogenous suspension. Do not vortex or agitate the beads.
6. Do not try to aspirate all of the liquid as this may result in loss ofbeads.
7. If beads are accidentally loaded onto the StageTip, the anti-body-conjugated to the beads also bind to C18. Using a higherpercentage of organic solvent for elution may result in theelution of these antibodies which will interfere with MS analy-sis. With 50 % ACN, the proteins do not elute.
8. Dissolve the lyophilized peptide digests with bRPLC solvent A.The solvent should be added in 1 ml aliquot each time. Soni-cate the tubes in water bath sonicator for 5 min to ensurecomplete dissolution of peptides.
9. 50 μl of 1 % Formic acid should be added to 96 wells prior tofraction collection to neutralize peptides.
Proteomic Strategies to Characterize Signaling Pathways 371
10. To ensure complete dissolution of the peptides, the tubes canbe kept on thermomixer set at 100�g for 20 min at 20 �Cto ensure complete dissolution.
11. TiO2 bead suspension in 5 % dihydrobenzoic acid turnsbrownish.
12. Alternatively, the tubes can be kept on thermomixer set at100�g for 15–30 min at 20 �C.
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