Top-down peptidomics of bodily fluids

Peptidomics 2014 Volume 1 47ndash64

AbbreviationsAD - Alzheimer DiseaseADPKD - autosomal dominant polycystic kidney diseaseaPRP - acidic proline-rich proteinAβ - Amyloid-βbPRP - basic proline-rich proteinCE - capillary electrophoresisCNS - central nervous systemCSF - cerebrospinal fluidESI - electrospray ionizationFPA - fibrinopeptide AHPLC - high permormance liquid chromatographyHst - HistatinIGF - Insulin like Growth FactorLC - liquid chromatographyLTQ - linear trap quadrupoleMALDI - matrix-assisted laser desorptionionizationMS - mass spectrometryPDR - proliferative diabetic retinopathypIgR - polymeric immunoglobulin receptorPTMs - post-translational modificationsQ-q-Q - triple quadrupoleSELDI - surface-enhanced laser desorptionionizationSgI - semenogelin ISgII - semenogelin IISJIA - systemic juvenile idiopathic arthritisSPE - solid phase extractionTOF - time of flightTβ10 - Thymosin β10

Tβ4 - Thymosin β4

1 IntroductionFunctioning of biological systems is the result of the dynamic interaction in the multiple scale of time and space of molecular networks interaction which in the case of proteins is strongly influenced by post-translational modifications (PTMs) In this field of particular interest are the proteolytic cleavages of bigger proteins leading to the production of naturally occurring peptides many

Abstract The naturally occurring peptides mainly arising from the proteolytic cleavage of larger proteins play several functions within the body (e g antihypertensive immuno-modulatory anti-microbial and antiviral mineral carriers) Their presence or the increase of their concentration could be connected to different pathologies and thereby some peptides could be useful biomarkers for the diagnosis or prognosis of the disease Peptidome research particularly within biological fluids therefore represents one of the most interesting and challenging purposes of proteomics In this review we describe the current state-of-the-art in peptidomics-based studies of several human bodily fluids (serum plasma urine cerebrospinal fluid saliva tears seminal fluid vitreous humor pancreatic juice) emphasizing the contribution of top-down proteomic platforms to the deep structural characterization of natural peptides and their post-translational modifications

Keywords Top-down proteomics serum plasma urine cerebrospinal fluid saliva tears seminal fluid vitreous humor pancreatic juice

Doi 102478ped-2014-0005received December 5 2013 accepted March 18 2014

Research Article Open Access

copy 2014 Claudia Martelli et al licensee De Gruyter Open This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 30 License

Claudia Martelli Federica Iavarone Federica Vincenzoni Tiziana Cabras Barbara Manconi Claudia Desiderio Irene Messana Massimo Castagnola

Top-down peptidomics of bodily fluids

Corresponding author Massimo Castagnola Istituto di Biochimica e Biochimica Clinica Facoltagrave di Medicina Universitagrave Cattolica del Sacro Cuore Largo F Vito 00168 Roma Italy Tel andor Fax ++39-06-3053598 E-mail massimocastagnolaicrmcnrit Claudia Martelli Federica Iavarone Federica Vincenzoni Istituto di Biochimica e Biochimica Clinica Facoltagrave di Medicina Universitagrave Cattolica del Sacro Cuore Roma ItalyTiziana Cabras Barbara Manconi Irene Messana Dipartimento di Scienze della Vita e delllsquoAmbiente Universitagrave di Cagliari Monserra-to (CA) ItalyClaudia Desiderio Massimo Castagnola Istituto di Chimica del Riconoscimento Molecolare ndash UOS Roma Consiglio Nazionale delle Ricerche Roma Italy These two Authors equally contributed to this manuscript

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48emsp emspClaudia Martelli et al

with biological activities different or even opposite to those of the parent protein One of the first surprising example is represented by the peptides derived from hemoglobin collectively termed hemorphins with opioid-like and other interesting activities [1-3] Nowadays it is increasingly evident that the information contained in the genes is not univocal and various functions may be played by the different gene-products originated by frameshift readings alternative splicing as well as by maturation of pre-pro-proteins and PTMs Indeed as demonstrated for hemoglobin many proteins hide within their structure the sequence of several biological active peptides that can be released according to precise temporal events often occurring after the lifespan of the parent protein The release of the latent substructures requires the coordination of a complex set of proteinases and the challenging comprehension of these phenomena is just at the beginning In vitro studies are often meaningless because cascades of fragmentation generated in vivo by specific molecular interactions cannot be reproduced with reliability in simplified artificial systems These fragmentation cascades cannot be investigated neither by bottom-up nor by middle-down proteomic platforms for their intrinsic limitation to discern the peptides naturally occurring from those generated in the sample by the digestion preceding the analytical step [4] The only viable strategy is the application of top-down proteomics which has the demanding purpose to elucidate the structure of intact proteinspeptides [5]

This review describes some results obtained for the characterization of human peptides in different bodily fluids by top-down platforms in particular underlying the studies devoted to the investigation of the roles and the potential application of the identified peptides The current huge amount of information obliged to select the most significant data reported in literature and we apologize for many relevant omissions

2 Blood plasma and serumBlood is a complex liquid tissue including a corpuscular and a non-corpuscular fraction and represents the major connection between cells and tissues of an organism allowing transport of both oxygen molecules (ie hormones amino acids carbohydrates lipids vitamins mineral salts and water) and metabolic waste products

The proteomepeptidome of a blood sample is directly linked to the metabolic state and can provide information on the physiological andor pathological processes occurring in the body The proteins and peptides present in serum

and plasma the non-corpuscular blood fractions are a combination of those playing circulatory functions and those secreted or released into the circulation from cells during both normal biological events and in diseases [6]

Being the most available clinical samples serum and plasma represent a convenient source of potential disease-biomarker peptides with minimal invasiveness Naturally occurring peptides typically derive from cleavages of the most abundant full-length proteins as a result of numerous proteases activity contained in these fluids or produced by cells [7 8]

As reported a high number of peptides is only present in serum and not detectable in plasma In fact more than 40 of the peptides detected in serum are generated by ex vivo processes during specimen collection and preparation introducing peptide signals which have to be interpreted with prudence [9] Serum generation is associated with coagulation cascade and activation of complement system These processes involve cell lysis and proteinases activation leading to the production of numerous peptides from proteins cleavages Moreover peptides are released from blood clot during the specimen collection [ie thymosin β4 (Tβ4) zyxin] In plasma preparation the addition of chelating agents deeply stabilized the proteinpeptide composition [9 10] However for several clinical analyses serum is preferred because anticoagulants can sometimes interfere with the results Whatever the fluid peptidomic analysis of serum andor plasma is critical due to the wide range of protein and peptide concentrations that spans over ten orders of magnitude [9] For this reason in 2002 the Human Plasma Proteome Project (HPPP) of the Human Proteome Organization started with the aims to standardize protocols of specimens collection handling and storage [9 10] stimulating the use of emerging technologies sharing results and creating global open-source databases repository [11 12]

Pre-treatment of serum and plasma samples for top-down peptidomic analysis is particularly critical due to the high complexity of these biological samples the large span of molecular weight distribution of peptides and the biological variability (ie gender age genetic environmental dietary and psychological factors) [13] Several pre-treatment strategies have been proposed for enrichment of blood peptides and removal of high abundance proteins i) centrifugal ultrafiltration at low speed and in denaturing conditions ii) protein precipitation with organic solvent (acetone or acetonitrile) and iii) cleaning-up with solid phase extraction (SPE) columns magnetic beads and disk plates All these procedures may or not be followed by a further fractionation step [SPE strategies gel electrophoresis



Top-down peptidomics of bodily fluids emsp emsp49

surface-derivatives chips or beads filtration with nanoporous substrates capillary electrophoresis (CE) and liquid chromatography (LC)] [14-19] An interesting recent paper compares methods designed to remove intact proteins from plasma in order to allow analysis of the peptidome by liquid chromatography-mass spectrometry (LC-MS) experiments [20] The authors introduced the use of Mass differential Tags for Relative and Absolute Quantification (mTRAQ) labeling coupled to Selected Reaction Monitoring assay to monitor each peptide and determine the peptide recovery thus evaluating the suitability of each method Other papers reported the development of mass spectrometry based quantitation methods for peptides analysis in serum [21] and plasma [16]

Small blood peptides are often the results of physiological or pathological processes within cell involving protease activities both as waste products or signal molecules they may hide a powerful role of diagnostic biomarker panel [7] Therefore many studies are devoted to identify and quantify blood peptidome patterns in particular for biomarker discovering

Richter et al [22] identified plasma peptides in extracts of human hemofiltrate generating a mass database and a sequence database with approximately 5000 peptides deriving from over 340 sequence entries of 75 different protein precursors 55 are plasma proteins [albumin fibrinogen A (RGD peptides) fibrinogen B α-1-microglobulin β-2-microglobulin zinc-α-2-glycoprotein α -2-HS-glycoprotein serum amyloid A protein haptoglobin profilin vitronectin desmocollin Tβ4 apolipoprotein C-III uteroglobin ubiquitin gelsolin somatomedin B hemopexin] 7 are peptide hormones [angiotensin 1 guanylin 22-115 uroguanylin 89-112 cardiodilatinatrial natriuretic peptide β-defensin 1 α-defensin 1 α-defensin 3 kininogen (light molecular weight chain)] cytokines growth factors and growth inhibitors [HCC-1 Insulin-like growth factor I (IGF-1) IGF-2 osteoinductive factor platelet derived growth factor osteopontin platelet basic protein pigment endothelium derived factor angiogenin I collagen XVIII] 33 belongs to complement factors [C3 C4A C9 D] transport proteins [transthyretin serotransferrin retinol binding protein transforming growth factor-binding protein IGF-binding protein] enzymes and enzyme inhibitors [lysozyme carboxypeptidase N pancreatic trypsin inhibitor cystatin C plasminogen α-2-antiplasmin inter-α-trypsin inhibitor complex component II α-1-antitrypsin hexokinase type II ribonuclease] and 5 are novel peptides

Amyloid-β (Aβ) peptides are among the most challenging peptides in blood because of their strict

relation with Alzheimer disease (AD) which is the most common neurodegenerative disorder As is largely known these peptides normally derive by the sequential cleavage of the amyloid precursor protein and when over-produced progressively aggregate as small neurotoxic oligomers in the brain leading to severe cortical dysfunction The Aβ variants circulating in plasma are Aβ1-40 and Aβ1-42 and it has been suggested that these peptides are transported into the brain via blood circulation [23] Many studies are focused on the development of effective analytical method for the detection of Aβ peptides [23 24] possible candidates as plasma biomarkers for prediction progression and therapeutic monitoring of the AD However to date the results are not completely clear and in some cases they donrsquot correlate each other [25 26] In addition to Aβ peptides Hu et al [27] reported that pancreatic polypeptide and B-type natriuretic peptide were strongly associated with the diagnosis of mild cognitive impairment and AD states Moreover plasma levels of these two peptides correlated with cerebrospinal fluid (CSF) Aβ1-42 levels and t-tauAβ1-42 ratio well-known CSF AD biomarkers and thus could serve as the basis of a plasma-based screening battery for AD

Another peptide found in blood that has multiple amazing functions is Tβ4 the main G-actin binding acetylated peptide present in almost any tissue and in extracellular media in mammals In blood it is present in the cytoplasm and nucleus of all cells (except erythrocytes) and in plasma and serum [28] It is surprising that a molecule as small as Tβ4 contains so many biological and structural functions such as wound healing stimulation of angiogenesis and suppression of inflammation [28] Its concentration in serum and in plasma corresponds to about 1 of the total amount of Tβ4 present in whole blood while its intracellular concentration in leukocytes including platelets is above 300 μM [29] In blood coagulation or ADP-induced platelets aggregation Tβ4 is released from platelets and partially cross-linked to fibrin Its linking to the fibrin clot might represent a mechanism to guarantee a high local concentration of the peptide at the site of the injury probably supporting subsequent wound healing processes [29] It has been shown that extracellular Tβ4 induces migration of cardiomyocytes and their survival as well [30] In addition to intact Tβ4 the N-terminal tetrapeptide (AcSDKP) and larger C and N-terminal fragments show several biological activities and are related to different pathological states [31-34] For instance serum samples from psoriasis patients exhibited altered levels of Tβ4 proteolytic peptides 1-17 22-43 and 25-45 [33] while plasma samples from patients with rheumatoid arthritis [34] showed that Tβ4 actin binding




site (LKKTETQ) was cleaved by the combined action of endoproteases and amminopeptidases to generate Tβ4 peptide ladders (fragment 19-43 20-43 22-43 23-43 24-43 25-43 26-43 30-43) In both cases the levels of proteolytic peptides are increased with respect to control samples and it has also been found that concentration of certain peptides was much higher in the disease states (ie fragment 25-43 and 22-43)

Many recent papers are focused on serumplasma peptidome patterns of patients affected by several cancer diseases [15] (ie brain tumors [35] thyroid carcinoma [36] pancreatic cancer [37 38] breast cancer [39] prostate cancer [40] multiple-myeloma and leukemia [41] and lung adenocarcinoma [42]) It has been suggested that different tumors secrete distinct proteases generating unique serum peptide profiles [40] In metastatic thyroid carcinoma [36] 12-peptide thyroid cancer signature was obtained with respect to control samples Ten of them had been previously assigned to other tumors [40] while one of the two newly identified peptides is a 54-amino acid-long fibrinogen-α fragment

In serum peptidome profiling of pancreatic cancer have been found decreased levels of platelet factor 4 with respect to normal samples [37] Platelet factor 4 is a member of C-X-C chemokine family and its decrease could suggest an enhancement of metalloproteinase activities due to their up-regulation A work conducted on pooled plasma samples from 15 breast cancer patients [39] showed that breast cancer and control samples had very similar sets of plasma proteins identified by the conventional bottom-up proteomic method but different degradation patterns obtained by top-down peptidomic analysis In particular breast cancer sample displayed 839 distinct peptides versus the 425 peptides detected in healthy controls Furthermore the degradation observed was substrate selective and for individual substrates the cleavage specificity could vary significantly (for instance in the case of apolipoprotein A-IV) [8] This finding suggests a significantly higher degradome activity in cancer and differences of the substrate proteolytic activity in the physiological and pathological states

In the lung adenocarcinoma serum peptidome profile 12 peptide candidates have been suggested as cancer biomarkers [42] Among them eight are reported as fibrinopeptide A (FPA) and FPA derived peptides (fragments 2-16 3-15 3-16 4-16 5-16 6-15 6-16 1-16) two belong to apolipoprotein A-IV (fragments 273-283 and 271-283) one to limbin (fragment 306-313) and one to amiloride-sensitive cation channel 4 (fragment 613-624)

In summary plasma and serum top-down peptidomic analysis could represent a powerful strategy for the

comprehension of biological processes such as signal transmission between cells circulation and transport of several molecules and development of diseases The identification of serumplasma biomarker diseases especially cancer disease is the first goal of all the research laboratories involved in the Plasma Proteome Project of the Human Proteome Organization [12]

3 UrineBecause of its easy non-invasive and non-painful collection large availability and stability urine is one of the most suitable bodily fluids for the analysis of peptides compared to other biological matrices

The urinary proteome is distributed among the soluble fraction the ldquosedimentsrdquo (principally consisting in epithelial cells and debris) the small membrane fragments and the ldquoexosomesrdquo (small vesicles excreted from cells facing the urine) [43] As a consequence of urine origin its peptidome is principally made up of small soluble peptides which derive naturally from plasma either excreted by several mechanisms from kidneys and urogenital tract or produced by proteolytic activity of extracellular proteases Therefore the peptidome and proteome of urine kidney and plasma are strictly correlated [44] Perturbation of urinary peptide fraction can occur in different disease states resulting in alteration of the urinary peptidomic profile andor increased urinary peptide excretion [45] In normal donors the protein content of urine is lower than 100 mgL however the urinary proteome and metabolome is highly variable as a consequence of different daily intake of fluids diets metabolic processes and circadian rhythms [46 47] Recently a new fingerprint database of human urinary proteome was developed to facilitate the use of this biofluid as a source of diagnostic biomarkers [48] The availability of large amount of sample allows easily operating an adequate peptide concentration for MS analysis without additional manipulation (top-down proteomic platform) The most employed techniques for urine peptidomic analysis by top-down approach are CE-MS [49-52] surface-enhanced laser desorptionionization (SELDI)-time of flight (TOF) MS [53] and matrix-assisted laser desorptionionization (MALDI) [54] also in association with metal microsphere peptide enrichment [55] and evaluating the important contribution of sample dilution for calculation of peptides relative abundance [56] Compared to LC-MS [57-59] these three techniques are more suitable for easy and fast analysis of low amount of samples (few microliters) Up to now over 2000 proteins and 100000 peptides have been detected in urine




[465860] and thanks to its easy manipulation and accessibility this biological fluid is one of the most used for biomarker discovery

The increase of urinary peptides levels is observed in patients with proteinuria [61] and up to the 20-30 of them originated from serum albumin fragmentation These peptides normally undetectable in healthy subjects could suggest the presence of an increased protease activity in these patients [62] The detection of proteinuriaalbuminuria is commonly used in clinics for the diagnosis of renal damage however these symptoms are not specific and frequently a late manifestations of the disease Therefore urinary biomarker peptides can allow together with the assessment of kidney damage also differential diagnosis of specific chronic renal diseases (diabetic nephropathy IgA nephropathy systemic vasculitis etc) and their progression status nephrological disorders (ie urotheliasis renal transplant rejection ureteropelvic junction obstruction cancer related to urogenital system) and non-nephrologicalurogenital pathologies (preeclampsia coronary artery disease heart failure stroke graft-versus-host disease etc) [57] CE-electrospray ionization (ESI)-TOF was applied for top-down peptidomic analysis of urine collected from individuals affected by different forms of chronic kidney diseases in comparison with control patient samples [49] In particular in chronic kidney disease the increase of serum proteins fragments (ie serum albumin fibrinogen and α1-antitrypsin hemoglobin α chain etc) and the decrease of diverse collagen fragments (especially from collagen α-1 chain) and kidney-proteins (ie uromodulin sodiumpotassium-transporting ATPase γ chain membrane-associated progesterone receptor component 1) were found In an another study involving patients affected by type 1 diabetes microalbuminuria and early renal failure [63] showed the urinary decrease of three peptides fragments of α-1 (IV) and α-1 (V) collagens and tenascin-X and the increase of three fragments of inositol pentakinophospate 2-kinase zona occludens 3 and FAT tumor suppressor 2 with respect to controls

CE-MS analysis of urine samples allowed also defining a specific peptide pattern for diagnosis and risk stratification in autosomal dominant polycystic kidney disease (ADPKD) [64] The developed diagnostic biomarker model was based on 142 peptides including markers previously associated with acute kidney injury and demonstrated to be highly specific for ADPKD

Ling et al [65] developed a top-down MS based method for a non invasive diagnosis of rejection of renal allografts MALDI-TOFTOF and linear trap quadrupole (LTQ)-Orbitrap analyses identified 40 urine peptides

specific of acute rejection and LC-ESI-triple quadrupole (Q-q-Q) in multiple-reaction monitoring scan mode allowed their quantification These peptides belonged to nine different proteins eight of collagen family (fragments of α-1 (I) α-1 (II) α-3 (I) α-4 (III) α-4(IV) α-4 (V) α-7 (I) α-18 (I) collagens) and one of uromodulin The analysis of the peptide sequences evidenced a specific proteolytic degradation of collagen and uromodulin during renal allografts acute rejection

Pegraverez and co-workers [66] studied the effect of the paracalcitol treatment on urinary peptidome in kidney transplant patient Paracalcitol a selective vitamin D receptor activator generally used in prevention and treatment of hyperparathyroidism was recently associated with improved prolonged survival in these patients Magnetic bead technology in coupling with MALDI-TOF fingerprinting mass spectrometry identified a significant alteration of the quantity of selected urinary peptides after 3 months of paracalcitol treatment useful for the elucidation of the possible molecular mechanism associated with drug administration

Many studies were devoted to the identification of urinary biomarkers for diagnosis disease progression recurrence and treatment response of cancer urogenital disease [67-70] Theodorescu et al [67] found a pattern of 22 polypeptides molecular masses as urothelial-carcinoma urinary biomarkers among them one of the most relevant is FPA In fact it has been demonstrated that the activation of coagulation pathways has a role in the preclinical phase of cancer and is associated with an increase of malignancy [71] The same research group discovered and validated urinary biomarkers for prostate cancer (fragments of collagen α-1 (III) chain collagen α-1 (I) chain psoriasis susceptibility 1 candidate gene 2 protein sodiumpotassium-transporting ATPase γ chain) [68] and for the prediction of non muscle invasive bladder cancer (fragments of uromodulin collagen α-1 (I) collagen α-1 (III) and membrane-associated progesterone receptor component 1) [69]

Very recently 86 urinary peptides (40 out of them sequenced) were associated with renal cell carcinoma as biomarker panel with a specificity of 87 [70] Urinary proteomic analysis was able to differentiate cholangiocarcinoma from primary sclerosing cholangitis and other benign biliary disorders by the identification of a panel of peptides mostly of them identified as fragments of interstitial collagens of both renal and extra-renal origins [72]

Significant variations of the peptidomic profile were also found in urine of patients with non-nephrologicalurogenital diseases Ling et al [73] identified a panel of 17




peptide biomarkers that could discriminate the different stages of systemic juvenile idiopathic arthritis (SJIA) The early diagnosis of this disease is difficult therefore delaying an appropriate therapy These peptides were identified as degradation products of eight different proteins α-1 antitrypsin collagen α-1 (I) (five peptides and three of them having overlapping sequences) collagen α-2 (I) collagen α-1 (III) (one peptide) collagen type α-2 (IX) (one peptide) fibrinogen α (two peptides having overlapping sequences) fibrinogen β (two peptides having overlapping sequences) and uromodulin (three peptides having overlapping sequences)

A peptide generated from the degradation of isoform 1 of fibrinogen α chain precursor able to differentiate between positive and negative Helicobacter pylori infected volunteers was identified in urine by MALDI-TOF and characterized by LTQ-Orbitrap tandem MS analysis [74]

Top-down proteomic analysis by MALDI-TOF was also applied to investigate the alteration induced by different altitudes (a model to explore diseases related to tissue hypoxia) in combination with Telmisartan pharmacological treatment [75] The results showed that the peptidome did not change with Telmisartan administration but only in relation to altitude exposure In particular in subjects with hypobaric hypoxia six different peptides have been detected differently expressed two of them belonging to uromodulin and to α-1 antitrypsin

Urinary proteomic analysis was able to discriminate patients with coronary artery disease from healthy individuals by the identification of a peptide biomarker panel including fragments of collagen type 1 and 3 fibrinogen α-chain sodiumpotassium transporting ATPase gamma chain α-1-antitrypsin granin-like neuroendocrine peptide precursor and membrane-associated progesterone receptor component 1 [76]

As mentioned till now urinary peptidomics is a useful tool for the individuation of molecular biomarkers of prognosis and diagnosis of several urogenital and non-urogenital diseases as deriving from specific proteolytic cleavages of both plasma and kidneys proteins However it is noteworthy to underline that some of the identified peptides biomarkers in urine are associated to different pathologies For instance the peptide DGApGKNGERGGpGGpGP [a degradation product of collagen α-1 (III)] is present both in urine of ADPKD and SJIA although the two diseases have different pathogenesis specifically ADPKD is an hereditary kidney disease caused by mutations in the PKD1 or the PKD2 genes while SJIA is a chronic inflammatory disease of childhood characterized by a combination of systemic symptoms [fever rash serositis (eg

pericarditis pleuritis)] and arthritis Further studies are surely necessary in order to establish validation clinical specificity and sensitivity of all the urinary biomarkers already identified

4 Cerebrospinal FluidThe cerebrospinal fluid (CSF) is a colorless liquid that surrounds the brain and the spinal cord It has several functions including mechanical protection metaboliteswaste products circulation and central nervous system (CNS) homeostasis regulation [77] CSF is secreted from several different components of the CNS in particular from the choroid plexus localized within lateral ventricles roof of the III ventricle and floor of the IV ventricle In the human ventricular system the CSF total volume is 125 ml with a daily production of 500 ml in adult and child while this value is significantly lower in newborns [78] CSF formation is the result of either blood ultrafiltration through the fenestrated capillaries endothelium and choroid epithelium of ependyma either brain tissue contribution and it is characterized by the presence of electrolytes glucose amino acids peptides proteins and metabolites Although the protein composition of CSF is very similar to blood the total protein concentration is about 200 times lower Furthermore as a consequence of the blood brain barrier filtration same components are present in a different ratio in the two biofluids resulting in a diverse distribution pattern of the most abundant proteins [77]

Due to the close contact with brain tissues CSF analysis offers an exclusive opportunity for studying CNS related diseases and biomarker discovering Particularly significant differences have been found in CSF proteome and peptidome in association to diverse CNS diseases indicating the high potential of CSF for biomarkers identification proteins and peptides usually absent in CSF can be present in a pathological state as a consequence of a blood brain barrier damage or to a local production (neurodegenerativetumoural diseases) [79]

The proteomic analysis of human CSF has been extensively reviewed [77 78] particularly focusing on neurodegenerative disease biomarker discovery [80] Being biomarkers mainly represented by small proteins and peptides (molecular weight less than 10 kDa) the top-down approach for CSF peptidomics is a successful strategy for identifying their candidates and studying CNS pathologies as following described

As for the other biofluids the conditions of storage and treatment of cerebrospinal fluid are crucial to




maintain the intact protein state for top-down proteomic analysis The immediate storage of the sample at -80degC andor the immediate treatment (ie centrifugation) are essential for preserving the original characteristics and inhibiting protease activity [81] A recent study evaluating the entity of protease activity during cerebrospinal fluid serum and plasma sample handling observed no significant activity of the endogenous enzymes during an appropriate time confirming the power of top-down proteomics for biomarker discovering and suggesting an intrinsic protective role of the sample from degradation [82] Nano-LC in coupling with Q-TOF-MS was used for peptidomic analysis of human lumbar CSF after removal of high molecular mass proteins and salts and solid phase cartridge enrichment focusing on identification of low molecular mass (lt5 kDa) peptides originating from in vivo enzymatic cleavage of large proteins [83] The top-down platform was successfully applied for the identification and sequencing of peptides with molecular mass lower than 3 kDa while trypsin digestion was necessary for the identification of bigger peptides and proteins Six different naturally occurring peptides belonging to fibrinogen αα-E chain precursor and three belonging to prothrombin granin-like neuroendocrine peptide and fibrinogen β-chain precursors respectively were characterized Another research [84] demonstrated the high potential of nanoESI-Q-TOF-MS instrumentation for characterization and sequencing of peptides within 9 kDa in human lumbar CSF and brain tissue with top-down approach after LC pre-fractionation and MALDI-TOF-MS identification following the procedure previously described for CSF high resolution peptide mapping [85] In this work Tβ4 the neuropeptide hormone NPY (43 kDa) and ubiquitin (85 kDa) were sequenced together with a series of peptides in the range 1-9 kDa belonging to 13 different precursor proteins ie α-1-antitrypsin albumin cholecystokinin chromogranin A fibrinogen α glyceraldehyde-3-phosphate-dehydrogenase haemoglobin β-chain neuroendocrine specific protein VGF osteopontin ProSAAS secretogranin I and II and transthyretin

The first study presenting an extensive proteomic and peptidomic characterization of human lumbar CSF on large scale appeared in 2008 [86] CSF was analyzed after dithiothreitol reduction and iodoacetamide alkylation followed by ultrafiltration the high molecular weight fraction (proteome) was studied by LC-MSMS in bottom-up approach following 1D gel separation and trypsin digestion while the low molecular one (peptidome) was directly analyzed by capillary LC- nano-ESI-LTQ-Orbitrap in top-down strategy using Higher-energy Collisional Dissociation fragmentation mode Several PTMs were

present on the characterized neuropeptides Most of the identified peptides were generated by cleavage of larger precursor proteins and probably for a specific biological activity purpose the C-terminal amidated joining peptide neurexophilin-3 and -4 peptides with pyroglutamic acid at N-terminal glutamine synaptogamins and phospholemman peptides with different PTMs cystein rich paragranulin-like peptide Insulin-like growth factor-2 glycosylated peptide glycosylated heparin-binding EGF-like growth factor and orexin-A peptides and polyproline peptides were some examples Several PTMs were present on the characterized neuropeptides A comprehensive study of CSF peptidomics appeared subsequently [87] Ultrafiltration procedures by molecular weight cut-off filters with or without previous treatments of CSF with acetonitrile andor formic acid were applied and compared for peptide identifications in the range 700-5000 Da by nanoLC off line coupled to MALDI-TOF-MS With respect to ultrafiltration only the treatment of CSF with 20 of acetonitrile resulted in an increased number of detected peptides from 2445 (untreated CSF) to 3543 Not considering the occurrence of PTMs tandem MS analysis identified 625 unique peptides sequences originating from 104 proteins

Most of the peptidomic analysis of CSF was devoted to study Aβ peptides in relation to neurodegenerative diseases To this regard the analysis of the peptides in their entire state is of importance for the identification of PTMs events that could be of relevance for studying and diagnosing neurodegenerative diseases CSF is an effective source of candidate peptide biomarkers for AD as reviewed in recent papers [25 88] Aβ peptides were in principle mainly analyzed by immune-based technologies SELDI-TOF-MS was applied to Aβ peptide characterization in CSF in relation to AD after protein chip affinity array [89] The comparison with Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) immunoblotting data established the high potential of SELDI-MS for expression profiling of the neurodegenerative disease the identified Aβ peptides showed different patterns in AD CSF with respect to controls and the exclusive presence of Aβ2-46 in AD sample was outlined Another study using the same procedure investigated Aβ peptides distribution in AD CSF sample and healthy control of the same range of age providing similar results [90]

Portelius et al described a MALDI-TOF-MS method in reflector mode [91 92] and nanoLC-LTQ-FT tandem MS [93] for Aβ peptides analysis in lumbar CSF after immunoprecipitation with monoclonal antibody bound to magnetic beads They identified N- and C-terminal




truncated Aβ peptides particularly Aβ1-16 Aβ1-33 Aβ1-39 and Aβ1-42 (some exhibiting oxidized methionine inside the sequence) showing a different pattern between pathological samples and healthy control In this work the use of isotopically labeled internal standard minimized the variation induced by sample preparation and analysis allowing the potential development of a future diagnostic tool MALDI-TOF-MS analysis of lumbar CSF from the same group of authors allowed distinguishing sporadic AD patients from normal and depressed individuals and familiar AD patients by the individuation of aberrant Aβ peptides pattern and isoforms [94] A novel separation and detection method for amino-terminal Aβ peptides variants in human CSF in association with MS identification after immunoprecipitation was also recently developed [95]

In a recent paper a new ultra performance LC method in coupling with Q-q-Q MS in Multiple Reaction Monitoring mode was developed and validated for the quantitative analysis of Aβ1-38 Aβ1-40 and Aβ1-42 in CSF after SPE [96] A CE method was also developed for analyzing five amyloid peptides of relevant clinical interest in CSF [97]

A novel procedure for analyzing CSF peptidome was recently described applying two schemes for the isolation of different peptide and protein fractions in post-mortem samples of AD patients [98] Low molecular weight peptides were analyzed in the soluble fraction after precipitation with a mixture of methanol and chloroform The pellet was re-solubilized and after reductionamidation re-precipitated the resulting supernatant was submitted to analysis of the peptides released from high molecular weight proteins The pellet was enzymatically digested for protein identification Both the peptide fractions were analyzed by MALDI-TOF-TOF and nanoLC in coupling with Ultra High Resolution-TOF-MS Peptides belonging to VGF nerve growth factor inducible complement C4 precursors and peptides of α-2-HS-glycoprotein were able to discriminate AD patients from controls Recently SELDI-TOF-MS was applied to CSF analysis of Aβ peptides in association to cognitive decline in sporadic AD and elderly schizophrenic patients showing a distinctive signature [99]

The proteomic analysis of lumbar CSF of amyotrophic lateral sclerosis (SLA) patients revealed interesting alterations with respect to controls allowing the identification of potential disease biomarker and evidencing the possible mechanisms involved The samples were analyzed by MALDI-TOF-MS after superparamagnetic particle isolation and by LC-ESI-LTQ tandem MS for peptides identification [100] Immunoprecipitation in coupling with mass spectrometry of undigested CSF was used to study the alterations of amyloid precursor protein

and Aβ peptide levels in multiple sclerosis patients also revealing at this level the counteraction of natalizumab treatment [101] Few papers deal with CSF peptidomics in relation to brain cancer diseases A recent review outlined the state of art of proteomic analysis applied to gliomas starting from different biological matrices including CSF However this study highlighted more bottom-up than top-down applications [102]

Top-down proteomic by our group recently identified in CSF a panel of bioactive peptides of hemoglobin origin including LVV- and VV-hemorphin-7 and other four peptides with sequence homology to α- and β-globin chains fragments as candidate biomarkers of prognosis of posterior cranial fossa pediatric brain tumor diseases [103] Ventricular CSF was collected intra-operatively and post-operatively from the same patients and analyzed by LC in coupling with high resolution ESI-LTQ-Orbitrap mass spectrometry in Data Dependent Scan after a simple pretreatment procedure outlining the importance of top-down proteomics A selective method for LVV- and VV-hemorphin-7 quantitation in CSF was also specifically developed and validated by CE-ESI-ion trap-MS starting from few biofluids volume after a simple sample pretreatment [104]

A biomarkers peptides panel of prospective diagnostic application of different neurodegenerative diseases was identified by peptidomic analysis of lumbar CSF in top-down approach by CE-TOF-MS [105] Of the about 1000 polypeptides measured 279 were identified by the combination of CE-MS data software elaboration and high resolution MS de novo sequencing

5 Saliva and gingival crevicular fluidHuman saliva is a bodily fluid secreted by three couples of major glands (parotid submandibular and sublingual glands) and by an individually variable number of minor salivary glands [106] The term ldquosalivardquo usually refers only to the fluid secreted by these glands and is the main issue of this section while the term ldquowhole salivardquo or ldquooral fluidrdquo refers to a more complex solution comprising oral exudates gingival crevicular fluid oral microflora desfoliating cells nasal secretion and food remnants [107] Peptides present in glandular saliva at first originate from a complex secretion process starting in acinar cells where many pre-pro-proteins are submitted to cleavages after the loss of the signal peptide generating a great number of smaller peptides [108] In some cases the process is exhaustive as for basic proline-rich proteins (bPRPs)




which are not detectable in saliva as pro-proteins [109] Acidic proline-rich proteins (aPRPs) on the contrary can be detected simultaneously as intact pro-proteins (PRP-1 PRP-2 PIF-s Db-s Pa as different possible isoforms) and as fragmentation products (PRP-3 PRP-4 PIF-f Db-f and the P-C peptide 44 aa residue long common to all aPRPs) [110] Glandular ldquosalivardquo contains some specific salivary peptide families too named histatins (Hst) statherins and PB peptide Histatins are a family of small histidine-rich peptides some of them with interesting antifungal activity [111] It is widely accepted that all the members of this family arise from two parent peptides Hst 1 and Hst 3 having very similar sequences and encoded by two genes (HIS1 and HIS2) Despite the very high sequence similarity these two peptides follow completely different PTM pathways [106] Hst 3 is submitted to a pre-secretory sequential cleavage generating at first Hst 6 (histatin 3 125) subsequently Hst 5 (histatin 3 124) and then other fragments [112] Hst 1 is not cleaved during secretion for the absence of a proper consensus sequence Hst 1 is phosphorylated on Ser-2 residue while Hst 3 is not due to the absence of a +2 flanking glutamic acid residue to Ser-2 essential for the kinase recognition Hst 1 is partly poly-sulfated on the four terminal tyrosines in submandibular glands differently from Hst 3 which lacks a tyrosine equivalent to Tyr-27 of Hst 1 probably essential for the tyrosylprotein sulfotransferase recognition [113] Statherin is an unusual tyrosine-rich phospho-peptide of 43 residues which should be related to the modulation of oral calcium concentration Many properties of P-B peptide suggest a functional relationship with statherin although no specific function of this peptide have been proposed to date [114] All this informations have been recently investigated by High Performance Liquid Chromatography (HPLC)-ESI-MS top-down platforms [115]

All the salivary peptides above reported are submitted after secretion to further cleavages generating a great number of different fragments Top-down HPLC-ESI-MS analysis revealed that the proteolytic cleavage generating these fragments occurred preferentially after a glutamine residue with a predominant specificity for a XPQ where X in the -3 flanking position is mainly represented by a lysine residue [116] Systematic evaluation of the enzyme activity responsible for these cleavages revealed the presence of a glutamine endoprotease exogenous in origin deriving from different Rhodia strains common hosts of dental plaque [117] The Rhodia enzymes have the ability to degrade gluten proteins and recent researches are trying to investigate if they can neutralize gluten epitopes [117]

Despite the efforts until now performed many questions on the functions of these peptides and their

fragments remain unresolved Statherin and aPRPs are surely involved in oral calcium homeostasis [106 107] but no hypothesis exists about the role of both the aPRPs fragmentation and P-C and P-B peptides Hst 3 and their fragments (especially Hst 5 and 6) display noticeable antifungal and antimicrobial activity [118] Surprisingly recent studies have demonstrated that even though its structural similarity with Hst 3 Hst 1 differently displayed interesting wound healing properties [119] N- to C- cyclization potentiated the peptide activity 1000-fold indicating that a specific peptide conformation was responsible for the effect [120] The minimally active domain was fragment 20ndash32 of the parent peptide Interestingly Hst 1 stimulated wound closure of primary cells of both oral and non-oral origin [121] which encouraged the therapeutic application of Hst 1-derived peptides in the treatment of different kinds of skin wound

Little information is available on the potential role of bPRPs and fragments except their hypothetical ability to protect against the harmful effects of tannins [122] However because recent studies have shown that full expression of bPRPs is reached after puberty [123] it is evident that they should exert age-linked functions For instance recent studies have demonstrated their potential role as mediators of bitter taste perception [124 125] Moreover some bPRPs fragments show potential anti-viral activity [126]

HPLC-ESI-MS top-down platforms have recently established that other naturally occurring fragments of bigger proteins are detectable in human whole saliva such as some fragments of glycerhaldeide-3-phosphate dehydrogenase and polymeric Ig receptor [115] but no further information is available about their significance in the oral cavity

Recent studies carried out with top-down platforms are indicating that some variations of the concentration of these peptides of their PTMs andor of naturally occurring fragments could be utilized as biomarker or clues of multifactorial diseases Anomalous fragment levels from different peptides (P-C P-B statherin histatins) were established in Type 1 (insulin dependent) diabetic patients [127] where a simultaneous decrease of the concentration of the parent peptides was also found suggesting a link between increased glucose concentration and oral microflora modification Hypo-phosphorylation of statherin Hst 1 and aPRPs (both entire and truncated isoforms) was found in a subset of patients with autism spectrum disorder [128] suggesting asynchronies in the activation of the pleitropic Golgi-casein kinase acting in the CNS during the fetal growth of these subjects All these studies are confirming that saliva characterized by




a non-invasive specimen collection is an attractive bodily fluid for diagnostic purposes with a particular concern to pediatric age [129]

A minor contribution to whole saliva is ensured by gingival crevicular fluid a transudate arising from the gingival plexus of gingival corium in minute quantities Top-down platforms have evidenced that this fluid contains high concentrations of interesting peptides such as α-defensins [130] Tβ4 and Tβ10 [131] The high amount of α-defensins probably originates from different leukocyte families transmigrating in high number in the gingival pocket while the origin of the high levels of β-thymosins is currently unknown Both peptides could modify their concentrations in several pathologies offering new opportunities for early diagnosis of local and systemic diseases by this fluid

6 Tears Human tear is a fluid secreted from the lachrymal gland with several important functions which beyond obvious humidification role include the prevention of infections and the ability to act as a barrier to the outside environment Only few studies have been devoted to the characterization of peptides in tears Nguyen-Khuong et al used an LC-MALDI-TOF-MS approach to show the presence of small molecular weight compounds in human tears but no structural identification was carried out [132] In another study performed on tears collected from patients suffering of xerophtalmia large quantities of the antimicrobial α-defensins 1ndash3 were detected by electrophoretic separation and characterization by in-gel tryptic digestionMALDI-TOF-MS analysis suggesting the occurrence of chronic inflammation in the ocular regions [133] Recently a top-down comprehensive study on the human reflex tear fluid [134] revealed the presence of a great variety of novel naturally occurring peptides derived from PRP4 and the polymeric immunoglobulin receptor (pIgR) PRP4 is a protein whose mRNA is abundantly expressed in lachrymal acinar cells also detected in submandibular von Ebnerrsquos sublingual and parotid glands [135] Many of the identified peptides showed an N-terminal pyroglutamate which as suggested by the authors could contribute to preserve their structure in the tear fluid rich in proteases [136] PIgR is an Fc receptor which facilitates the transcytosis of dimeric IgA and IgM to the apical surface During epithelial transcytosis pIgR is submitted to cleavage at the level of the extracellular domain and the cleaved pIgR peptides are released either in a free form or associated with IgA or IgM [137] Free

peptides have been shown to possess innate antimicrobial properties [138] and it has been also demonstrated that Fc receptors derived peptides show cytokine-like activity [139] In this respect it should be outlined that recent studies proved that the tear fluid contains chemosignal compounds [140]

7 Miscellaneous

71 Seminal fluid

Seminal plasma is an acellular fluid providing a safe medium for spermatozoa The ejaculate consists of up to 10 spermatozoa and 90 seminal plasma Less than 1 of seminal plasma derives from urethral and bolbourethral (Cowperrsquos) glands 2-5 derives from testes including the epididymis and vasa deferentia whereas the 25-30 and 65-75 are of prostate and seminal vesicles respectively [141 142] The secretion of the prostate represents the most important source of acid phosphatase citric acid inositol calcium magnesium and zinc present in the ejaculate Seminal vesicles contain fructose ascorbic acid and prostaglandins Epididymal secretions are rich in L-carnitine and neutral α-glucosidase while a small amount of the seminal fructose originates from the ampulla of the ductus deferens [143] The composition of the secretions from the different glands is used in the clinic to evaluate male fertility Human seminal plasma is also a rich source of proteins that have important roles in the capacitation of the spermatozoa modulation of the immune responses of the uterus formation of the tubal sperm reservoir [144 145] and lastly in both the sperm-zona pellucida interaction and sperm-egg fusion [146 147] The diverse biomolecules content of seminal plasma allows the successful fertilization of the oocyte by the spermatozoa

Immediately after the ejaculation semen spontaneously coagulates into a semi-solid gelatinous mass due to the interactions between semenogelin I and II (SgI and SgII) the major coagulating proteins in human semen [148-150]

Without proper semen liquefaction men are frequently subfertile [151] The liquefaction of semen is a physiological process mainly connected to degradation of SgI and SgII by a prostate-derived kallikrein-like serine protease called prostate-specific antigen although other trypsin-like proteases originating from the prostate such as human kallikrein hK2 prostatic acid phosphatase transglutaminase and prostatin also contribute to the degradation of SgI and SgII at alternative sites [150 152-154]




Fung et al characterized over 30 peptide components within unfractionated seminal plasma by means of MALDI-TOF-MS and LC-ESI-MSMS approach the majority of which were found to be proteolytic products of either SgI or SgII [155]

It has been demonstrated that SgI and SgII and their related degradation peptides show biological functions such as antibacterial activity [156-158] or α-inhibin-like activity [159 160] Recently some studies correlated naturally occurring peptides released by physiological cleavage of semen coagulum with the enhancement of HIV infection [161-165]

72 Vitreous Humor

The human vitreous humor is the transparent highly-hydrated gel which occupies the posterior segment of the eye between the lens and the retina and it makes up about 80 of the volume of the eyeball [166]

It is composed almost entirely of water (99) with the remainder consisting of a mixture of collagen fibers hyaluronic acid hyalocytes inorganic salts and lipids [167]

The protein composition consists largely of albumin and other components like globulins coagulation proteins complement factors and low-molecular-weight proteins [168] The accumulation of proteins in the vitreous could be due to local secretion (eg glycoprotein) blood filtration (eg albumin) or diffusion from the surrounding tissues [169] Since vitreous is in contact with the retina physiological and pathological conditions of the retina itself could affect the protein and peptides composition within this fluid especially when the blood-retinal barrier is disrupted [170] Evidences show that changes of specific vitreous proteins are associated with various vitreoretinal diseases [171-173] and particularly in diabetic macular edema [174] or retinopathy [175] Although several studies have been carried out with the aim to identify proteins differentially expressed in various pathologies mainly by using techniques such as 2D electrophoresis coupled to MALDI-TOF 2D electrophoresis coupled to LC-MSMS [176 177] or Difference Gel Electrophoresis (DIGE) [178 179] only few information is available about peptide composition of the vitreous humor Rolling et al [180] found significantly higher concentrations of atrial natriuretic peptide in vitreous humor of patients affected by active proliferative diabetic retinopathy (PDR) with respect to quiescent PDR and diabetes (without PDR) patients or controls by applying a specific and sensitive radioimmunoassay They speculated that atrial natriuretic peptide a 28-amino acid peptide hormone with natriuretic

and vasodilatory properties [181] contributes to the fibrovascular complications of PDR through possible direct antiangiogenic effects in the eye No information is available on naturally occurring peptides detected by top-down analysis

73 Pancreatic juice

Proteomic analysis of pancreatic juice was mainly focused on the research of chronic pancreatitis and pancreatic cancer biomarkers In fact as proximal bodily fluid this juice is rich in proteins that are shed directly from the pancreas

A priori pancreatic juice should contain proteins from the zymogen granule content plasma proteins from leak or transcytosis into ductal fluid proteins shed from the apical membrane of acinar and ductal cells and intracellular proteins released because of damage either occurring in pathogenic states or during the collection process The pancreatic juice is a not easily accessible bodily fluid and it is primarily collected during surgery or by endoscopic retrograde cholangio-pancreatography Due to its collection invasiveness few data are available about control subjects The normal pancreatic juice proteome is therefore not yet fully understood Up to date only one study was carried out by Doyle et al [182] on normal pancreatic juice collected by retrograde cholangio-pancreatography from 3 females showing clinical abdominal symptoms but no apparent pancreatic pathology after investigation Proteins were analyzed by a bottom-up approach losing the majority of the information about naturally occurring peptides The largest group of proteins recognized was obviously digestive enzymes and plasma proteins Studies to define proteins and peptides organellar localization proteolysis processes translocation and PTMs with a top-down platform are necessary to provide more in-depth knowledge Several studies were performed to characterize and quantify proteins and peptides in pancreatic juice of patients affected by pancreatitis and pancreatic cancer In a preliminary work the proteins and peptides separation was carried out by means of 2-D electrophoresis followed by MALDI-TOF MS proteome characterization [183-186]

Groslashnborg et al [187] used Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) followed by LC-MSMS for the proteomic analysis of pancreatic juice (collected during surgery) from 3 patients with pancreatic adenocarcinoma In addition to expected pancreatic proteins they found a number of lsquolsquocancer-associatedrsquorsquo proteins including annexin 5 Carcinoembryonic antigen-related cell adhesion molecule 5 and several S100 proteins




Chen et al [188] compared pancreatic juice from a chronic pancreatitis patient to a pooled sample from 10 lsquolsquonormalrsquorsquo subjects which have suffered of pancreatitis Quantitation was carried out by using Isotope-Coded Affinity Tags (ICAT) labeling followed by 2-D LC-MSMS Proteins up-regulated in chronic pancreatitis included fibrinogen β chain plasminogenplasmin Neural cell adhesion molecule L1 serum albumin α 1b-glycoprotein and α 2-macroglobulin Some of these however have also been reported to be up-regulated in pancreatic cancer using the same approach [189]

Several other studies were carried out for a comparative andor quantitative analysis for biomarker discovery in pancreatic juice [190 191] but completely lacked the issue of evaluating peptides without the constraint of assuming tryptic digestion All this data should be confirmed and better understood using a top-down platform and a label-free quantization of natural occurring peptides and proteins

8 Conclusions As also outlined in the present review peptides being involved in key regulatory processes play a central role in many physiological processes in living systems Nevertheless in the last twenty years the studies devoted to a comprehensive characterization of proteins and peptides in a tissue cell or bodily fluid have been dominated by the throughput bottom-up techniques which are intrinsically not suitable for the characterization of naturally occurring peptides As a result significantly fewer ldquopeptidomicsrdquo studies with respect to the ldquoproteomicsrdquo ones are present in the literature Even few these studies demonstrated that important information can be obtained by the analysis of this subset of the proteome For instance the peptidomic characterization of bodily fluids provided valuable hints about the role played by specific proteinases under different physio-pathological conditions [127] and disclosed new naturally occurring proteolytic cascades [112] The consideration that processing patterns of peptides can be used for diagnostic purposes has represented an important stimulus for the exploration of endogenous peptides under pathological conditions

On the whole the results described in this review demonstrate that the discovery of relevant biomarker candidates and drug targets is possible with top-down platform often aided by bottom-up strategies and other enzymatic and chemical treatments Given the rapid development in all the major technical aspects and the increasingly widespread application of top-down

platforms it is expected that peptidomics will give in the near future an important contribution in the field of drug discovery and biomarker characterization

Acknowledgements The authors acknowledge the financial support of Cagliari University Catholic University of Rome MIUR Italian National Research Council (CNR) Regione Sardegna and Nando Peretti Foundation according to their programs of scientific diffusion

The authors have declared no conflicts of interest

References[1] Giardina B Messana I Scatena R Castagnola M The

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[2] Zhao Q Garreau I Sannier F Piot J M Opioid peptides derived from hemoglobin hemorphins Biopolymers 1997 43 75-98

[3] Nyberg F Sanderson K Glaumlmsta E L The hemorphins a new class of opioid peptides derived from the blood protein hemoglobin Biopolymers 1997 43 147-156

[4] Messana I Cabras T Iavarone F Vincenzoni F Urbani A Castagnola M Unraveling the different proteomic platforms J Sep Sci 201336 128-139

[5] Tran J C Zamdborg L Ahlf D R Lee J E Catherman A D Durbin K R et al Mapping intact protein isoforms in discovery mode using top-down proteomics Nature 2011 480 254-258

[6] Omenn G S Menon R Adamski M Blackwell T Haab B B Gao W et al The Human Plasma and Serum Proteome in V Thongboonkerd (Ed) Proteomics of human body fluids Humana Press Totowa New Jersey 2007 10

[7] Liotta L A Ferrari M Petricoin E Clinical proteomics written in blood Nature 2003 425 905

[8] Shen Y Liu T Tolić N Petritis B O Zhao R Moore R J et al Strategy for degradomic-peptidomic analysis of human plasma J Proteome Res 9 2339-2346

[9] Tammen H Schulte I Hess R Menzel C Kellmann M Mohring T et al Peptidomic analysis of human blood specimens Comparison between plasma specimens and serum by differential peptide display Proteomics 2005 5 3414-3422

[10] Tammen H Hess R Collection and handling of blood specimens for peptidomics Methods Mol Biol 2011 728 151-159

[11] Omenn G S States D J Adamski M Blackwell T W Menon R Hermjakob H et al Overview of the HUPO Plasma Proteome Project results from the pilot phase with 35 collaborating laboratories and multiple analytical groups generating a core dataset of 3020 proteins and a publicly-available database Proteomics 2005 5 3226-3245

[12] Rai A J Gelfand C A Haywood B C Warunek D J Yi J Schuchard M D et al HUPO Plasma Proteome Project specimen collection and handling towards the standardization




of parameters for plasma proteome samples Proteomics 2005 5 3262-3277

[13] Villanueva J Philip J Chaparro C A Li Y Toledo-Crow R DeNoyer L et al Correcting Common Errors in Identifying Cancer-Specific Serum Peptide Signatures J Proteome Res 2005 4 1060-1072

[14] Luque-Garcia J L Neubert T A Sample preparation for serumplasma profiling and biomarker identification by mass spectrometry J Chromatogr A 2007 1153 259-276

[15] Petricoin EF Belluco C Araujo R P Liotta L A The blood peptidome another dimension of information content for cancer biomarker discovery Nature Rev Cancer 2006 6 961ndash967

[16] DrsquoImperio M Della Corte A Facchiano A Di Michele M Ferrandina G et al Standardized sample preparation phases for a quantitative measurement of plasma peptidome profiling by MALDI-TOF J Proteomics 2010 73 1355-1367

[17] Aristoteli L P Molloy M P Baker M S Evaluation of endogenous plasma peptide extraction methods for mass spectrometric biomarker discovery J Proteome Res 2007 6 571-581

[18] Tanaka K Tsugawa N Kim Y O Sanuki N Takeda U L J Lee A new rapid and comprehensive peptidome analysis by one-step direct transfer technology for 1-D electrophoresisMALDI mass spectrometry Biochem Biophys Res Commun 2009 379 110-114

[19] Hansen H G Overgaard J Lajer M Hubalek F Hoslashjrup P Pedersen L et al Finding diabetic nephropathy biomarkers in the plasma peptidome by high-throughput magnetic bead processing and MALDI-TOF-MS analysis Proteomics Clin Appl 2010 4 697-705

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[21] Bakun M Karczmarski J Poznanski J Rubel T Rozga M Malinowska A et al An integrated LC-ESI-MS platform for quantitation of serum peptide ladders Application for colon carcinoma study Proteomics Clin Appl 2009 3 932-946

[22] Richter R Schulz-Knappe P Schrader M Staumlndker L Juumlrgens M Tammen H et al Composition of the peptide fraction in human blood plasma database of circulating human peptides J Chromatogr B Biomed Sci Appl 1999 726 25-35

[23] Varesio E Rudaz S Krause K-H Veuthey J-L Nanoscale liquid chromatography and capillary electrophoresis coupled to electrospray mass spectrometry for the detection of amyloid-β peptide related to Alzheimerrsquos disease J Chromatogr A 2002 974 135-142

[24] Picou R A Kheterpal I Wellman A D Minnamreddy M Ku G Gilman S D Analysis of Aβ (1-40) and Aβ (1-42) monomer and fibrils by capillary electrophoresis J Chromatogr B Analyt Technol Biomed Life Sci 2011 879 627-632

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[28] Crockford D Turjman N Allan C Angel J Thymosin beta4 structure function and biological properties supporting current and future clinical applications Ann N Y Acad Sci 2010 119 179-189

[29] Mannherz H G Hannappel E The β-thymosins intracellular and extracellular activities of a versatile actin binding protein family Cell Motil Cytoskel 2009 66 839-851

[30] Bock-Marquette I Saxena A White M D Dimaio J M Srivastava D Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration survival and cardiac repair Nature 2004 432 466-472

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[32] Hannappel E Thymosin β4 and its posttranslational modifi-cations Ann N Y Acad Sci 2010 1194 27-35

[33] Plavina T Hincapie M Wakshull E Subramanyam M Hancock W S Increased plasma concentrations of cytoskeletal and Ca2+-binding proteins and their peptides in psoriasis patients Clin Chem 2008 54 1805-1814

[34] Zheng X Wu S L Hincapie M Hancock W S Study of the human plasma proteome of rheumatoid arthritis J Chromatogr A 2009 1216 3538-3545

[35] Villanueva J Philip J Entenberg D Chaparro C A Tanwar M K Holland E C et al Serum peptide profiling by magnetic particle-assisted automated sample processing and MALDI-TOF mass spectrometry Anal Chem 2004 76 1560ndash1570

[36] Villanueva J Martorella A J Lawlor K Philip J Fleisher M Robbins R J et al Serum Peptidome Patterns That Distinguish Metastatic Thyroid Carcinoma from Cancer-free Controls Are Unbiased by Gender and Age Mol Cell Proteomics 2006 5 1840ndash1852

[37] Fiedler G M Leichtle A B Kase J Baumann S Ceglarek U Felix K et al Serum Peptidome Profiling Revealed Platelet Factor 4 as a Potential Discriminating Peptide Associated with Pancreatic Cancer Clin Cancer Res 2009 15 3812-3819

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[39] Shen Y Tolicacute N Liu T Zhao R Petritis B O Gritsenko M A et al Blood Peptidome-Degradome Profile of Breast Cancer PLoS ONE 2010 5(10) e13133 doi101371journalpone0013133

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[49] Good D M Zuumlrbig P Argileacutes A Bauer H W Behrens G Coon J J et al Naturally Occurring Human Urinary Peptides for Use in Diagnosis of Chronic Kidney Disease Mol Cell Proteomics 2010 9 2424ndash2437

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[51] Stalmach A Albalat A Mullen W Mischak H Recent advances in capillary electrophoresis coupled to mass spectrometry for clinical proteomic applications Electro-phoresis 2013 34 1452ndash1464

[52] Latosinska A Frantzi M Vlahou A Mischak H Clinical applications of capillary electrophoresis coupled to mass spectrometry in biomarker discovery Focus on bladder cancer Proteomics Clin Appl 2013 7 779-793

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[54] Fiedler G M Baumann S Leichtle A Oltmann A Kase J Thiery J et al Standardized Peptidome Profiling of Human Urine by Magnetic Bead Separation and Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass Spectrometry Clin Chem 2007 53 421ndash428

[55] Zhao M Deng C Zhang X Yang P Facile synthesis of magnetic metal organic frameworks for the enrichment of low-abundance peptides for MALDI-TOF MS analysis Proteomics 2013 13 3387-3392

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[59] Sauvage F-L Gastinel L N Marquet P Untargeted screening of urine peptides with liquid chromatography coupled to hybrid linear-ion trap mass spectrometry J Chromatogr A 2012 1259 138-147

[60] Kentsis A Monigatti K Dorff K Campagne F Bachur R Steen H Urine proteomics for profiling of human disease using high accuracy mass spectrometry Proteomics Clin Appl 2009 3 1052-1061

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[63] Merchant M L Perkins B A Boratyn G M Ficociello L H Wilkey D W Barati M T et al Urinary Peptidome May Predict Renal Function Decline in Type 1 Diabetes and Microalbu-minuria J Am Soc Nephrol 2009 20 2065ndash2074

[64] Kistler A D Serra A L Siwy J Poster D Krauer F Torres V E et al Urinary Proteomic Biomarkers for Diagnosis and Risk Stratification of Autosomal Dominant Polycystic Kidney Disease A Multicentric Study PLoS ONE 2013 8(1) e53016 doi101371journalpone0053016

[65] Ling X B Sigdel T K Lau K Ying L Lau I Schilling J et al Integrative Urinary Peptidomics in Renal Transplantation Identifies Biomarkers for Acute Rejection J Am Soc Nephrol 2010 21 646ndash653

[66] Peacuterez V Saacutenchez A Bayeacutes B Navarro-Muntildeoz M Lauzurica R Pastor M C Romero R Effect of Paricalcitol on the Urinary Peptidome of Kidney Transplant Patients Transplant Proc 2010 42 2924ndash2927

[67] Theodorescu D Wittke S Ross M M Walden M Conaway M Just I et al Discovery and validation of new protein biomarkers for urothelial cancer a prospective analysis Lancet Oncol 2006 7 230ndash240

[68] Theodorescu D Schiffer E Bauer H W Douwes F Eichhorn F Polley R et al Discovery and validation of urinary biomarkers for prostate cancer Proteomics Clin Appl 2008 2 556ndash570

[69] Schiffer E Vlahou A Petrolekas A Stravodimos K Tauber R Geschwend J et alPrediction of Muscle-invasive Bladder Cancer Using Urinary Proteomics Clin Cancer Res 2009 15 4935-4943

[70] Franzti M Metzger J Banks R E Husi H Klein J Dakna M et al Discovery and validation of urinary biomarkers for detection of renal cell carcinoma J Proteomics 2014 98 44-58

[71] Miller G J Bauer K A Howarth D J Cooper J A Humphries S E Rosenberg R D Increased incidence of neoplasia of the digestive tract in men with persistent activation of the coagulant pathway J Thromb Haemost 2004 2 2107ndash2114

[72] Metzger J Negm A A Plentz R R Weismuumlller T J Wedemeyer J Karlsen T H et al Urine proteomic analysis differentiates cholangiocarcinoma from primary sclerosing




cholangitis and other benign biliary disorders Gut 2013 62 122-130

[73] Ling X B Lau K Deshpande C Park J L Milojevic D Macaubas C et al Urine Peptidomic and Targeted Plasma Protein Analyses in the Diagnosis and Monitoring of Systemic Juvenile Idiopathic Arthritis Clin Proteom 2010 6 175ndash193

[74] Xiao D Meng F L He L H Gu Y X Zhang J Z Analysis of the urinary peptidome associated with Helicobacter pylori infection World J Gastroenterol 2011 17 618-624

[75] Mainini V Gianazza E Chinello C Bilo G Revera M Giuliano A et al Modulation of urinary peptidome in humans exposed to high altitude hypoxia Mol Biosyst 2012 8 959-66

[76] Delles C Schiffer E von Zur Muhlen C Peter K Rossing P Parving H H et al Urinary proteomic diagnosis of coronary artery disease identification and clinical validation in 623 individuals J Hypertens 2010 28 2316-2322

[77] Ramstrom M Bergquist J Proteomics of Human Cerebrospinal Fluid in V Thongboonkerd (Ed) Proteomics of Human Body Fluids Principles Methods and Applications Humana Press Inc Totowa NJ 2007 12

[78] Yuan X Desiderio D M Proteomics analysis of human cerebrospinal fluid J Chromatogr B Analyt Technol Biomed Life Sci 2005 815 179-189

[79] Ekegren T Hanrieder J Bergquist J Clinical perspectives of high-resolution mass spectrometry-based proteomics in neuroscience exemplified in amyotrophic lateral sclerosis biomarker discovery research J Mass Spectrom 2008 43 559-571

[80] Kroksveen A C Opsahl J A Aye T T Ulvik RJ Berven F S Proteomics of human cerebrospinal fluid discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics J Proteomics 2011 74 371-388

[81] Simonsen AH Bahl JM Danborg PB Lindstrom V Larsen SO Grubb A et al Pre-analytical factors influencing the stability of cerebrospinal fluid proteins J Neurosci Methods 2013 215 234-240

[82] Pesek J Kruumlger T Krieg N Schiel M Norgauer J Groszligkreutz J et al Native chromatographic sample preparation of serum plasma and cerebrospinal fluid does not comprise a risk for proteolytic biomarker loss J Chromatogr B 2013 923-924 102-109

[83] Yuan X Desiderio D M Human cerebrospinal fluid peptidomics J Mass Spectrom 2005 40 176-181

[84] Moumlhring T Kellmann M Juumlrgens M Schrader M Top-down identification of endogenous peptides up to 9 kDa in cerebrospinal fluid and brain tissue by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry J Mass Spectrom 2005 40 214-226

[85] Heine G Zucht HD Schuhmann MU Buumlrger K Juumlrgens M Zumkeller M et al High-resolution peptide mapping of cerebrospinal fluid a novel concept for diagnosis and research in central nervous system diseases J Chromatogr B Analyt Technol Biomed Life Sci 2002 782 353-361

[86] Zougman A Pilch B Podtelejnikov A Kiehntopf M Schnabel C Kumar C et al Integrated analysis of the cerebrospinal fluid peptidome and proteome J Proteome Res 2008 7 386-399

[87] Houmllttauml M Zetterberg H Mirgorodskaya E Mattsson N Blennow K Gobom J Peptidome analysis of cerebrospinal

fluid by LC-MALDI MS PLoS ONE 2012 7(8) e42555 doi101371journalpone0042555

[88] Zuumlrbig P Jahn H Use of proteomic methods in the analysis of human body fluids in Alzheimer research Electrophoresis 2012 33 3617-3630

[89] Lewczuk P Esselmann H Meyer M Wollscheid V Neumann M Otto M et al The amyloid-beta (Abeta) peptide pattern in cerebrospinal fluid in Alzheimerrsquos disease evidence of a novel carboxyterminally elongated Abeta peptide Rapid Commun Mass Spectrom 2003 17 1291-1296

[90] Maddalena A S Papassotiropoulos A Gonzalez-Agosti C Signorell A Hegi T Pasch T et al Cerebrospinal fluid profile of amyloid beta peptides in patients with Alzheimerrsquos disease determined by protein biochip technology Neurodegener Dis 2004 1 231-235

[91] Portelius E Westman-Brinkmalm A Zetterberg H Blennow K Determination of beta-amyloid peptide signatures in cerebrospinal fluid using immunoprecipitation-mass spectrometry J Proteome Res 2006 5 1010-1016

[92] Portelius E Zetterberg H Andreasson U Brinkmalm G Andreasen N Wallin A et al An Alzheimerrsquos disease-specific beta-amyloid fragment signature in cerebrospinal fluid Neurosci Lett 2006 409 215-9

[93] Portelius E Tran AJ Andreasson U Persson R Brinkmalm G Zetterberg H et al Characterization of amyloid beta peptides in cerebrospinal fluid by an automated immunopreci-pitation procedure followed by mass spectrometry J Proteome Res 2007 6 4433-4439

[94] Portelius E Andreasson U Ringman JM Buerger K Daborg J et al Distinct cerebrospinal fluid amyloid beta peptide signatures in sporadic and PSEN1 A431E-associated familial Alzheimerrsquos disease Mol Neurodegener 2010 5 2 httpwwwmolecularneurodegenerationcomcontent512

[95] Hauszligmann U Jahn O Linning P Janszligen C Liepold T Portelius E et al Analysis of amino-terminal variants of amyloid-β peptides by capillary isoelectric focusing immunoassay Anal Chem 2013 85 8142-8149

[96] Lame M E Chambers E E Blatnik M Quantitation of amyloid beta peptides Aβ(1-38) Aβ(1-40) and Aβ(1-42) in human cerebrospinal fluid by ultra-performance liquid chroma-tography-tandem mass spectrometry Anal Biochem 2011 419 133-139

[97] Mesbah K Verpillot R de Lrsquoescaille F Falmagne J B Taverna M Contribution of CE to the analysis of protein or peptide biomarkers Methods Mol Biol 2013 984 167-190

[98] Wijte D McDonnell L A Balog C I Bossers K Deelder AM Swaab D F et al A novel peptidomics approach to detect markers of Alzheimerrsquos disease in cerebrospinal fluid Methods 2012 56 500-507

[99] Albertini V Benussi L Paterlini A Glionna M Prestia A Bocchio-Chiavetto L et al Distinct cerebrospinal fluid amyloid-beta peptide signatures in cognitive decline associated with Alzheimerrsquos disease and schizophrenia Electrophoresis 2012 33 3738-3744

[100] von Neuhoff N Oumeraci T Wolf T Kollewe K Bewerunge P Neumann B et al Monitoring CSF proteome alterations in amyotrophic lateral sclerosis obstacles and perspectives in translating a novel marker panel to the clinic PLoS ONE 2012 7 e44401 doi101371journalpone0044401




[101] Augutis K Axelsson M Portelius E Brinkmalm G Andreasson U Gustavsson MK et al Cerebrospinal fluid biomarkers of β-amyloid metabolism in multiple sclerosis Mult Scler 2013 19 543-552

[102] Kalinina J Peng J Ritchie J C Van Meir E G Proteomics of gliomas initial biomarker discovery and evolution of technology Neuro Oncol 2011 13 926-492

[103] Desiderio C DrsquoAngelo L Rossetti D V Iavarone F Giardina B Castagnola M et al Cerebrospinal fluid top-down proteomics evidenced the potential biomarker role of LVV- and VV-hemorphin-7 in posterior cranial fossa pediatric brain tumors Proteomics 2012 12 2158-2166

[104] Zeccola M Longhi R Rossetti D V DrsquoAngelo L Tamburrini G Di Rocco C et al Development and validation of a capillary electrophoresis tandem mass spectrometry analytical method for the determination of Leu-Val-Val- and Val-Val-hemorphin-7 peptides in cerebrospinal fluid J Chromatogr A 2012 1267 170-177

[105] Jahn H Wittke S Zuumlrbig P Raedler T J Arlt S Kellmann M et al Peptide fingerprinting of Alzheimerrsquos disease in cerebrospinal fluid identification and prospective evaluation of new synaptic biomarkers PLoS One 2011 6 e26540 doi101371journalpone0026540

[106] Messana I Inzitari R Fanali C Cabras T Castagnola M Facts and artifacts in proteomics of body fluids What proteomics of saliva is telling us J Sep Sci 2008 31 1948-1963

[107] Helmerhorst E J Oppenheim F G Saliva a dynamic proteome J Dent Res 2007 86 680-693

[108] Messana I Cabras T Pisano E Sanna M T Olianas A Manconi B et al Trafficking and postsecretory events responsible for the formation of secreted human salivary peptides a proteomics approach Mol Cell Proteomics 2008 7 911-926

[109] Messana I Cabras T Inzitari R Lupi A Zuppi C Olmi C et alCharacterization of the human salivary basic proline-rich protein complex by a proteomic approach J Proteome Res 2004 3 792-800

[110] Inzitari R Cabras T Onnis G Olmi C Mastinu A Sanna M T et al Different isoforms and post-translational modifications of human salivary acidic proline-rich proteins Proteomics 2005 5 805-815

[111] Oppenheim F G Xu T McMillian F M Levitz S M Diamond R D Offner G D et al Histatins a novel family of histidine-rich proteins in human parotid secretion Isolation characterization primary structure and fungistatic effects on Candida albicans J Biol Chem 1988 263 7472-7477

[112] Castagnola M Inzitari R Rossetti D V Olmi C Cabras T Piras V et al A cascade of 24 histatins (histatin 3 fragments) in human saliva Suggestions for a pre-secretory sequential cleavage pathway J Biol Chem 2004 279 41436-41443

[113] Cabras T Fanali C Monteiro J A Amado F Inzitari R Desiderio C et al Tyrosine polysulfation of human salivary histatin 1 A post-translational modification specific of the submandibular gland J Proteome Res 2007 6 2472-2480

[114] Inzitari R Cabras T Rossetti D V Fanali C Vitali A Pellegrini M et al Detection in human saliva of different statherin and P-B fragments and derivatives Proteomics 2006 6 6370-6379

[115] Castagnola M Cabras T Iavarone F Vincenzoni F Vitali A Pisano E et al Top-down platform for deciphering the human salivary proteome Matern Fetal Neonatal Med 2012 25 27-43

[116] Helmerhorst E J Sun X Salih E Oppenheim F G Identi-fication of Lys-Pro-Gln as a novel cleavage site specificity of saliva-associated proteases J Biol Chem 2008 283 19957-19966

[117] Zamakhchari M Wei G Dewhirst F Lee J Schuppan D Oppenheim F G et al Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract PLoS One 2011 6 e24455 doi101371journalpone0024455

[118] Kavanagh K Dowd S Histatins antimicrobial peptides with therapeutic potential J Pharm Pharmacol 2004 56 285-289

[119] Oudhoff M J Bolscher J G Nazmi K Kalay H van lsquot Hof W Amerongen A V et al Histatins are the major wound-closure stimulating factors in human saliva as identified in a cell culture assay FASEB J 2008 22 3805-3812

[120] Oudhoff M J Kroeze K L Nazmi K van den Keijbus P A van lsquot Hof W Fernandez-Borja M et al Structure-activity analysis of histatin a potent wound healing peptide from human saliva cyclization of histatin potentiates molar activity 1000-fold FASEB J 2008 22 3805-3812

[121] Oudhoff M J van den Keijbus P A Kroeze K L Nazmi K Gibbs S Bolscher J G et al Histatins enhance wound closure with oral and non-oral cells J Dent Res 2009 88 846-850

[122] Bennick A Interaction of plant polyphenols with salivary proteins Crit Rev Oral Biol Med 2002 13 184-196

[123] Cabras T Pisano E Boi R Olianas A Manconi B Inzitari R et al Age-dependent modifications of the human salivary secretory protein complex J Proteome Res 2009 8 4126-4134

[124] Cabras T Melis M Castagnola M Padiglia A Tepper B J Messana I et al Responsiveness to 6-n-propylthiouracil (PROP) is associated with salivary levels of two specific basic proline-rich proteins in humans PLoS One 2012 7 e30962 doi101371journalpone0030962

[125] Melis M Aragoni M C Arca M Cabras T Caltagirone C Castagnola M et al Marked increase in PROP taste respon-siveness following oral supplementation with selected salivary proteins or their related free amino acids PLoS One 2013 8 e59810 doi101371journalpone0059810

[126] Robinovitch M R Ashley R L Iversen J M Vigoren E M Oppenheim F G Lamkin M Parotid salivary basic proline-rich proteins inhibit HIV-I infectivity Oral Dis 2001 7 86-93

[127] Cabras T Pisano E Mastinu A Denotti G Pusceddu P P Inzitari R et al Alterations of the salivary secretory peptidome profile in children affected by type 1 diabetes Mol Cell Proteomics 2010 9 1099-1108

[128] Castagnola M Messana I Inzitari R Fanali C Cabras T Morelli A et al Hypo-phosphorylation of salivary peptidome as a clue to the molecular pathogenesis of autism spectrum disorders J Proteome Res 2008 7 5327-5332

[129] Castagnola M Cabras T Vitali A Sanna M T Messana I Biotechnological implications of the salivary proteome Trends Biotechnol 2011 29 409-418

[130] Pisano E Cabras T Montaldo C Piras V Inzitari R Olmi C et al Peptides of human gingival crevicular fluid determined by HPLC-ESI-MS Eur J Oral Sci 2005 113 462-468




[131] Inzitari R Cabras T Pisano E Fanali C Manconi B Scarano E et al HPLC-ESI-MS analysis of oral human fluids reveals that gingival crevicular fluid is the main source of oral thymosins beta(4) and beta(10) J Sep Sci 2009 32 57-63

[132] Nguyen-Khuong T Fitzgerald A Zhao Z Willcox M Walsh B J Improvements for the visualization of low-molecular weight protein and peptides of human tears using MALDI Proteomics 2008 8 3424ndash3432

[133] Lo LndashH Wu PndashC Wu Yndash C Shiea J Characterization of human neutrophil peptides (α-defensins) in the tears of dry eye patients Anal Methods 2010 2 1934-1940

[134] Hayakawaa E Landuyt B Baggerman G Cuyvers R Lavigne R Luytene W et al Peptidomic analysis of human reflex tear fluid Peptides 2013 42 63-69

[135] Dickinson D P Thiesse M A major human lacrimal gland mRNA encodes a new proline-rich protein family member Invest Ophthalmol Vis Sci 1995 36 2020ndash2031

[136] de Souza G A Godoy L M F Mann M Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors Genome Biol 2006 7 R72 httpgenomebiologycom200678R72

[137] Phalipon A Corthesy B Novel functions of the polymeric Ig receptor well beyond transport of immunoglobulins Trends Immunol 2003 24 55ndash58

[138] Phalipon A Cardona A Kraehenbuhl J P Edelman L Sansonetti P J Corthesy B Secretory component a new role in secretory IgA-mediated immune exclusion in vivo Immunity 2002 17 107ndash115

[139] Saxon A Ke Z Bahati L Stevens R H Soluble CD23 containing B cell supernatants induce IgE from peripheral blood B-lymphocytes and costimulate with interleukin-4 in induction of IgE J Allergy Clin Immunol 1990 86 333ndash344

[140] Gelstein S Yeshurun Y Rozenkrantz L Shushan S Frumin I Roth Y et al Human tears contain a chemosignal Science 2011 331 226ndash230

[141] Duncan M W Thompson H S Proteomics of semen and its constituents Proteomics Clin Appl 2007 1 861-875

[142] Rodriacuteguez-Martiacutenez H Kvist U Ernerudh J Sanz L Calvete J J Seminal plasma proteins what role do they play Am J Reprod Immunol 2011 66 11-22

[143] Owen D H Katz D F A review of the physical and chemical properties of human semen and the formulation of a semen stimulant J Androl 2005 26 459-469

[144] Evans J P Kopf G S Molecular mechanisms of sperm-egg interaction and egg activation Andrologia 1998 30 297-307

[145] Jansen S Ekhlasi-Hundrieser M Toumlpfer-Petersen E Sperm adhesion molecules structure and function Cells Tissues Organs 2001 168 82-92

[146] Primakoff P Myles D G Penetration adhesion and fusion in mammalian sper-egg interaction Science 2002 296 2183-2185

[147] Yi Y J Mananhdar G Oko R J Bredd W G Sutovsky P Mechanism of sper-zona pellucid penetration during mammalian fertilization 26S proteasome as a candidate egg coat lysine Soc Reprod Fertil Suppl 2007 63 385-408

[148] de Lamirande E Semenogelin the main protein of the human semen coagulum regulates sperm function Semin Thromb Hemost 2007 33 60-68

[149] Lilja H Oldbring J Rannevik G Laurell C B Seminal vesicle-secreted proteins and their reactions during gelation

and liquefaction of human semen J Clini Invest 1987 80 281-285

[150] Peter A Lilja H Lundwall A Malm J Semenogelin I and semenogelin II the major gel-forming proteins in human semen are substrates for transglutaminase Eur J Biochem 1998 252 216-221

[151] Mann T Lutwak-Mann C Male Reproductiove Function and Semen Themes and trends in Physiology Biochemestry and Investugative andrology Springer-Verlag Berlin Heidelberg New York 1981

[152] Tauber P F Zaneveld L J D Propping D Schumacher G F B Components of human split ejaculates II Enzymes and proteinase inhibitors J Reprod Fertil 1976 46 165-171

[153] Brillard-Bourdet M Rehault S Juliano L Ferrer M Moreau T Gauthier F Amydolytic activity of prostatic acid phosphatise on human semenogelin and semenogelin-derived synthetic substrates Eur J Biochem 2002 269 390-395

[154] Christensson A Laurell C B Lilja H Enzymatic activity of postate-specific antigen and its reactions with extracellular serine proteinase inhibitor Eur J Biochem 1990 194 755-763

[155] Fung K Y C Glode L M Green S Duncam M W A comprehensive Characterization of the Peptide and Protein Constituents Of Human Seminal Fluid Prostate 2004 61 171-181

[156] Zhao H Lee W Shen J Li H Zhang Y Identification of novel semenogelin I-derived antimicrobial peptide from liquefied human seminal plasma Peptides 2008 29 505-511

[157] Bourgeon F Evrard B Brillard-Bourdet M Colleu D Jeacutegou B Pineau C Antibacterial activity of human seminal plasma Biol Reprod 2004 70 768-774

[158] Edstroumlm A M Malm J Frohm B Martellini J A Giwercman A Moumlrgelin M et al The Major Bactericidal Activity of Human Seminal Plasma Is Zinc-Dependent and Derived from Fragmentation of the Semenogelins J Immunol 2008 181 3413-3421

[159] Seidah N G Ramasharma K Sairam M R Chreacutetien M Partial amino acid sequencing of human seminal plasma peptide with inhibin-like activity FEBS Lett 1984 167 98-102

[160] Ramasharma K Sairam M R Seidah N G Chreacutetien M Manjunath P Sciller P E et al Isolation structure and synthesis of a human seminal plasma peptide with inhibin-like activity Science 1984 223 1199-1202

[161] Muumlnch J Ruumlcker E Staumlndker L Adermann K Goffinet C Schindler M et al Semen-derived amyloid fibrils drastically enhance HIV infection Cell 2007 131 1059-1071

[162] Doncel G F Joseph T Thurman A R Role of semen in HIV-1 transmission inhibitor or facilitator Am J Reprod Immunol 2011 65 292-301

[163] Martellini J A Cole A L Svoboda P Stuchlik O Chen L M Chai K X et al HIV-1 Enhancing effect of Prostatic Acid Phosphatase Peptides Is Reduced in Human seminal Plasma PloS One 2011 6 e16285 doi101371journalpone0016285

[164] Roan N R Muumlller J A Liu H Chu S Arnold F Stuumlrzel C et al Peptides Released by Physiological Cleavage of Semen Coagulum Proteins Form Amyloids that Enhance HIV Infection Cell Host Microbe 2011 10 541-550




[165] Arnold F Schnell J Zirafi O Stuumlrzel C Meier C Weil T et al Naturally Occurring Fragments from Two Distinct Regions of the Prostatic Acid Phosphatase Form Amyloidogenic Enhancers of HIV Infection J Virol 2012 86 1244-1249

[166] Bishop P N Structural macromolecules and supramolecular organisation of the vitreous gel Prog RetinEye Res 2000 19 323ndash344

[167] Sebag J The vitreous in WHart (Ed) Physiology of the Eye Adlers Mosby St Louis 1992 7

[168] Ulrich J N Spannagl M Kampik A Gandorfer A Components of the fibrinolytic system in the vitreous body in patients with vitreoretinal disorders Clin Experiment Ophthalmol 2008 36 431ndash436

[169] Wu C W Sauter J L Johnson P K Chen C D Olsen TW Identification and localization of major soluble vitreous proteins in human ocular tissue Am J Ophthalmol 2004 137 655-661

[170] Shitama T Hayashi H Noge S Uchio E Oshima K Haniu H et al Proteome profiling of vitreoretinal diseases by cluster analysis Proteomics Clin Appl 2008 2 1265-1280

[171] de Boer J H van Haren M A de Vries-Knoppert W A Baarsma G S de Jong P V Postema F J et al Analysis of IL-6 levels in human vitreous fluid obtained from uveitis patients patients with proliferative intraocular disorders and eye bank eyes Curr Eye Res 1992 11 181ndash186

[172] Funatsu H Yamashita H Ikeda T Mimura T Eguchi S Hori S Vitreous levels of interleukin-6 and vascular endothelial growth factor are related to diabetic macular edema Ophthalmology 2003 110 1690ndash1696

[173] Hattenbach L O Allers A Gumbel H O Scharrer I Koch F H Vitreous concentrations of TPA and plasminogen activator inhibitor are associated with VEGF in proliferative diabetic vitreoretinopathy Retina 1999 19 383ndash389

[174] Funatsu H Yamashita H Nakamura S Mimura T Eguchi S Noma H et al Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor are related to diabetic macular edema Ophthalmology 2006 113 294-301

[175] Kim T Kim S J Kim K Kang U B Lee C Park K S et al Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients Proteomics 2007 7 4203ndash4215

[176] Shitama T Hayashi H Noge S Uchio E Oshima K Haniu H et al Proteome Profiling of Vitreoretinal Diseases by Cluster Analysis Proteomics Clin Appl 2008 2 1265-1280

[177] Ouchi M West K Crabb J W Kinoshita S Kamei M Proteomic analysis of vitreous from diabetic macular edema Exp Eye Res 2005 81 176-182

[178] Garciacutea-Ramiacuterez M Canals F Hernaacutendez C Colomeacute N Ferrer C Carrasco E et al Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE) a new strategy for identifying potential candidates

in the pathogenesis of proliferative diabetic retinopathy Diabetologia 2007 50 1294ndash1303

[179] Wang H Feng L Hu J W Xie C L Wang F Characterisation of the vitreous proteome in proliferative diabetic retinopathy Proteome Sci 2012 10 15 httpwwwproteomescicomcontent10115

[180] Rollin R Mediero A Martiacutenez-Montero J C Roldaacuten-Pallareacutes M Suaacuterez-Leoz M Vidal-Fernaacutendez P et al Atrial natriuretic peptide in the vitreous humor and epiretinal membranes of patients with proliferative diabetic retinopathy Mol Vis 2004 10 450-457

[181] Levin E R Gardner D G Samson W K Natriuretic peptides N Engl J Med 1998 339 321-328

[182] Doyle C J Yancey K Pitt H A Wang M Bemis K Yip-Schneider M T et al The proteome of normal pancreatic juice Pancreas 2012 41 186-194

[183] Tian M Cui Y Z Song G H Zong M J Zhou X Y Chen Y et al Proteomic analysis identifies MMP-9DJ-I and AIBG as overexpressed proteins in pancreatic juice from pancreatic ductal adenocarcinoma patients BMC Cancer 2008 8 241 httpwwwbiomedcentralcom1471-24078241

[184] Lv S Gao J Zhu F Li Z Gong Y Xu G et al Transthyretin identified by proteomics is overabundant in pancreatic juice from pancreatic carcinoma and originates from pancreatic islets Diagn Cytopathol 2011 39 875-881

[185] Zhou L Lu Z Yang A Deng R Mai C Sang X et al Comparative proteomic analysis of humanpancreatic juice methodological study Proteomics 2007 7 1345-1355

[186] Gao J Zhu F Lv S Li Z Ling Z Gong Y et al Identification of pancreatic juice proteins as biomarkers of pancreatic cancer Oncol Rep 2010 23 1683-1692

[187] Groslashnborg M Bunkenborg J Kristiansen T Z Jensen O N Yeo C J Hruban R H et al Comprehensive proteomic analysis of human pancreatic juice J Proteome Res 2004 3 1042-1055

[188] Chen R Pan S Cooke K Moyes K W Bronner M P Goodlett D R et al Comparison of pancreas juice proteins from cancer versus pancreatitis using quantitative proteomic analysis Pancreas 2007 34 70ndash79

[189] Chen R Pan S Yi E C Donohoe S Bronner M P Potter J D et al Quantitative proteomic profiling of pancreatic cancer juice Proteomics 2006 6 3871ndash3879

[190] Chen R Pan S Duan X Nelson B H Sahota R A de Rham S et al Elevated level of anterior gradient-2 in pancreatic juice from patients with pre-malignant pancreatic neoplasia Mol Cancer 2010 9 149 httpwwwmolecular-cancercomcontent91149

[191] Makawita S Smith C Batruch I Zheng Y Ruumlckert F Gruumltzmann R et al Integrated proteomic profiling of cell line conditioned media and pancreatic juice for the identification of pancreatic cancer biomarkers Mol Cell Proteomics 2011 10 M111008599




with biological activities different or even opposite to those of the parent protein One of the first surprising example is represented by the peptides derived from hemoglobin collectively termed hemorphins with opioid-like and other interesting activities [1-3] Nowadays it is increasingly evident that the information contained in the genes is not univocal and various functions may be played by the different gene-products originated by frameshift readings alternative splicing as well as by maturation of pre-pro-proteins and PTMs Indeed as demonstrated for hemoglobin many proteins hide within their structure the sequence of several biological active peptides that can be released according to precise temporal events often occurring after the lifespan of the parent protein The release of the latent substructures requires the coordination of a complex set of proteinases and the challenging comprehension of these phenomena is just at the beginning In vitro studies are often meaningless because cascades of fragmentation generated in vivo by specific molecular interactions cannot be reproduced with reliability in simplified artificial systems These fragmentation cascades cannot be investigated neither by bottom-up nor by middle-down proteomic platforms for their intrinsic limitation to discern the peptides naturally occurring from those generated in the sample by the digestion preceding the analytical step [4] The only viable strategy is the application of top-down proteomics which has the demanding purpose to elucidate the structure of intact proteinspeptides [5]

This review describes some results obtained for the characterization of human peptides in different bodily fluids by top-down platforms in particular underlying the studies devoted to the investigation of the roles and the potential application of the identified peptides The current huge amount of information obliged to select the most significant data reported in literature and we apologize for many relevant omissions

2 Blood plasma and serumBlood is a complex liquid tissue including a corpuscular and a non-corpuscular fraction and represents the major connection between cells and tissues of an organism allowing transport of both oxygen molecules (ie hormones amino acids carbohydrates lipids vitamins mineral salts and water) and metabolic waste products

The proteomepeptidome of a blood sample is directly linked to the metabolic state and can provide information on the physiological andor pathological processes occurring in the body The proteins and peptides present in serum

and plasma the non-corpuscular blood fractions are a combination of those playing circulatory functions and those secreted or released into the circulation from cells during both normal biological events and in diseases [6]

Being the most available clinical samples serum and plasma represent a convenient source of potential disease-biomarker peptides with minimal invasiveness Naturally occurring peptides typically derive from cleavages of the most abundant full-length proteins as a result of numerous proteases activity contained in these fluids or produced by cells [7 8]

As reported a high number of peptides is only present in serum and not detectable in plasma In fact more than 40 of the peptides detected in serum are generated by ex vivo processes during specimen collection and preparation introducing peptide signals which have to be interpreted with prudence [9] Serum generation is associated with coagulation cascade and activation of complement system These processes involve cell lysis and proteinases activation leading to the production of numerous peptides from proteins cleavages Moreover peptides are released from blood clot during the specimen collection [ie thymosin β4 (Tβ4) zyxin] In plasma preparation the addition of chelating agents deeply stabilized the proteinpeptide composition [9 10] However for several clinical analyses serum is preferred because anticoagulants can sometimes interfere with the results Whatever the fluid peptidomic analysis of serum andor plasma is critical due to the wide range of protein and peptide concentrations that spans over ten orders of magnitude [9] For this reason in 2002 the Human Plasma Proteome Project (HPPP) of the Human Proteome Organization started with the aims to standardize protocols of specimens collection handling and storage [9 10] stimulating the use of emerging technologies sharing results and creating global open-source databases repository [11 12]

Pre-treatment of serum and plasma samples for top-down peptidomic analysis is particularly critical due to the high complexity of these biological samples the large span of molecular weight distribution of peptides and the biological variability (ie gender age genetic environmental dietary and psychological factors) [13] Several pre-treatment strategies have been proposed for enrichment of blood peptides and removal of high abundance proteins i) centrifugal ultrafiltration at low speed and in denaturing conditions ii) protein precipitation with organic solvent (acetone or acetonitrile) and iii) cleaning-up with solid phase extraction (SPE) columns magnetic beads and disk plates All these procedures may or not be followed by a further fractionation step [SPE strategies gel electrophoresis


















































































7 Miscellaneous

71 Seminal fluid









72 Vitreous Humor





73 Pancreatic juice
























































































































































































































































































































7 Miscellaneous

71 Seminal fluid









72 Vitreous Humor





73 Pancreatic juice















































































































































































































































































































7 Miscellaneous

71 Seminal fluid









72 Vitreous Humor





73 Pancreatic juice




































































































































































































































































































7 Miscellaneous

71 Seminal fluid









72 Vitreous Humor





73 Pancreatic juice
























































































































































































































































































7 Miscellaneous

71 Seminal fluid









72 Vitreous Humor





73 Pancreatic juice











































































































































































































































































7 Miscellaneous

71 Seminal fluid









72 Vitreous Humor





73 Pancreatic juice



































































































































































































































































7 Miscellaneous

71 Seminal fluid









72 Vitreous Humor





73 Pancreatic juice
























































































































































































































































7 Miscellaneous

71 Seminal fluid









72 Vitreous Humor





73 Pancreatic juice














































































































































































































































7 Miscellaneous

71 Seminal fluid









72 Vitreous Humor





73 Pancreatic juice












































































































































































































































72 Vitreous Humor





73 Pancreatic juice


















































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































Date post:	27-Nov-2023
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Top-down peptidomics of bodily fluids

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