Exploring the Hidden Human Urinary Proteome via Ligand Library

Exploring the Hidden Human Urinary Proteome via Ligand Library Beads Annalisa Castagna,†,⊥ Daniela Cecconi,‡,⊥ Lau Sennels,§ Juri Rappsilber,§ Luc Guerrier,| Frederic Fortis,| Egisto Boschetti,| Lee Lomas,| and Pier Giorgio Righetti*,‡ University of Verona, Department of Clinical and Experimental Medicine, Unit of Internal Medicine, and Department of Agricultural and Industrial Biotechnologies, Verona, Italy, FIRC Institute for Molecular Oncology Foundation (IFOM), Milano, Italy, and Ciphergen Biosystems Inc, Fremont, California 94555, United States of America Received May 27, 2005

The human urinary proteome has been reassessed and re-evaluated via a novel concentration/ equalization technique, exploiting beads coated with hexameric peptide ligand libraries. These beads act by capturing the whole protein spectra contained in the sample, by drastically reducing the level of the most abundant species, while strongly concentrating the more dilute and rare ones. In a control urine sample, 134 unique proteins could be identified. The first bead eluate (in thiourea, urea, and CHAPS) permitted the identification of 317 gene products, whereas the second eluate (in 9 M urea, pH 3.8) allowed the identification of another 95 unique proteins. By eliminating redundancies, a total of 383 unique gene products could be identified in human urines. This represents a major increment as compared to data reported in recent literature. By comparing our data with those reported to the present, an additional 251 proteins could be added to the list, thus bringing the total unique gene products so far identified in human urines to ca. 800 species. Keywords: proteomics • dynamic range • ligand libraries • urinary proteins • hidden proteome • biochips

Introduction For decades now, clinical chemistry research has been focused on finding, in any tissue specimen but especially in body fluidssplasma, urine, tears, lymph, seminal plasma, milk, saliva, spinal fluidsnew indicators or markers for disease. This search for biomarkers is particularly appealing in body fluids, since their collection is minimally invasive or, in the case of urine, noninvasive. Yet, even bodily fluids are not immune from severe problems that have so far hampered the discovery of novel markers, e.g., both plasma and serum exhibit tremendous variations in individual protein abundances, typically of the order of 1010 or more, with the result that, in any typical twodimensional (2-D) map, only the high-abundance proteins are displayed.1-3 In the case of urine, the problems are further aggravated by their very low protein content requiring a concentration step of 100- to 1000-fold, coupled with its high * To whom correspondence should be addressed. Prof. Pier Giorgio Righetti, Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Via Mancinelli 7, Milano 20131, Italy. Tel: +39-02-23993082.Fax: +39-02-23993080.E-mail: [email protected]. † University of Verona, Department of Clinical and Experimental Medicine, Unit of Internal Medicine, Verona, Italy. ‡ University of Verona, Department of Agricultural and Industrial Biotechnologies, Verona, Italy. § FIRC Institute for Molecular Oncology Foundation (IFOM). | Ciphergen Biosystems Inc. ⊥ A. Castagna and D. Cecconi contributed equally and share the first authorship. 10.1021/pr050153r CCC: $30.25

 2005 American Chemical Society

salt levels, requiring their concomitant removal prior to any analytical step.4 Notwithstanding these severe problems, scientists are still working on the project of the “human protein index”,5 by which clinicians and pathologists had been envisioned standing at the patient’s bedside and consulting (via satellite, of course) a huge database containing all polypeptide spots of any possible tissue and body fluid, with the aim of finding qualitative and/ or quantitative changes in any of these spots, correlated with the insurgence of a given disease, a dream that, after a more than twenty year-long march, might not be too far from fruition.6,7 Since serum is thought to contain most, if not all, human proteins (perhaps in the order of millions, if one includes also distinct clonal immunoglobulin sequences),3 most efforts have been devoted to mining this vast body of polypeptide chains, potentially able to contain all possible markers of diseases. The results, over 25 years of research, have been impressive: from a few dozen proteins identified (mostly by immuno-blots) in the seventies,8 to 325 distinct proteins,2 up to an impressive list of 1175 distinct gene products.3 Quite disappointingly, however, although a number of interesting candidate marker proteins have been proposed,3 no new assays for diseases have been recently approved by the FDA. In the past few years, considerable research efforts have been devoted to mapping the human urinary proteome, since this is perhaps the only one that can be collected in a fully noninvasive manner and in large volumes repeatedly over a Journal of Proteome Research 2005, 4, 1917-1930

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research articles period of time and for a considerable period of time. A flurry of papers have appeared outlining high throughput methods for preparing large quantities suitable for 2-D mapping and also attempting at establishing near-standard proteomic maps for clinical-chemical purposes.9-19 Although the vast majority of them have exploited 2-D maps, a few reports have also described 1-D and 2-D chromatographic approaches.9,18,19 Moreover, 2-D map analysis has been already exploited in different clinical settings such as bladder cancer,20,21 Bence Jones proteinuria,22,23 rheumatoid arthritis,24 urinary tract infections and glomerular or nonglomerular diseases,25,26 chronic exposure to cadmium,27 characterization of urinary apolipoproteins and monitoring adaptive changes in unilateral nephrectomy28 and searching for novel candidate markers for prostatic cancer.29 Most of these approaches require a large number of steps for urine preparation prior to 2-D mapping, such as precipitation with protamine sulfate, removal of glycosaminoglycans, several dialysis steps, lyophilization, gel filtration, even immuno-subtraction of the most abundant proteins and other pre-fractionation tools.30 All of these steps render proteomic analysis of human urines quite cumbersome and time-consuming, and this without even taking into consideration the severe sample losses occurring at each sample handling step.11,30 A novel approach for mining the “unseen proteome” that exploits solid phase ligand libraries was recently described.31-33 The library is constituted of polymeric chromatographic beads, each bead carrying a unique hexapeptide ligand at a local concentration similar to classical affinity sorbents (e.g., about 50 µmol/mL of swollen beads). The number of different ligands (hence different types of affinity beads) dictated by the combinatorial synthesis approach is of several dozen of million. Since the number of different ligands from the library is significantly larger than the total number of proteins predicated in the initial mixture, theoretically each protein may find its corresponding ligand. The technology of this ligand library is based on the pioneering work of Merrifield34 on solid-phase synthesis, and using the “split, couple, recombine” method, libraries of potentially billions of different peptide ligands can be created. Thus, due to this tremendous ligand diversity, within the library there is theoretically a ligand for every protein, antibody, peptide, etc., present in the starting material. This unique approach is based on a different philosophy in regard to the problem of discovering the hidden proteome: instead of simplifying the complex mixture into fractions, or partitioning away the most abundant species, it captures all the species present in solution up to the saturation of the solid phase ligand library. This automatically results in a dramatic dilution of the most abundant species, with a concurrent concentration of the dilute and rare ones. Here, the concept is applied to the analysis of very diluted urinary proteins that normally escape to regular detection methods. The results suggest that we were quite far from completing the proteomic profiling of urine and that a lot more remained to be discovered.

Materials and Methods Chemicals. Urea, thiourea, tributylphosphine (TBP), glycine, sodium dodecyl sulfate (SDS), and 3-[3-cholamidopropyl dimethylammonio]-1-propanesulfonate (CHAPS) were obtained from Fluka Chemie (Buchs, Switzerland). Bromophenol blue, agarose, and Pharmalytes were from Pharmacia-LKB (Uppsala, Sweden). Acrylamide, N′,N′-methylenebisacrylamide, 1918

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ammonium persulfate, N,N,N′,N′,tetramethyl-ethylenediamine (TEMED), dithiothreitol (DTT), Sypro Ruby, Silverquest as well as the linear Immobiline dry strips pH gradient 3-10 (7 cm long) were from Bio-Rad Laboratories (Hercules, CA). Ethanol, methanol, glycerol, sodium hydroxide, hydrochloric acid, acetone, and acetic acid were from Merck (Darmstadt, Germany). Molecular marker kit, colloidal Coomassie blue and 16% acrylamide-tricine gels were from Invitrogen (Carlsbad, CA). Sample Collection and Preparation. Urines from 8 apparently healthy volunteers (age 24-26 yr, four males and four females) were collected after informed consent, immediately chilled on ice and processed as follows. After measuring the volume (200 mL per person) the pH was adjusted to a value of 7.0 with 1 N NaOH, then four “Complete protease inhibitor cocktail” tablets (Roche Diagnostics, Barkeley, USA) were added, to inhibit the activity of endogenous proteases present in the specimen. Urines were centrifuged at 4 °C for 30 min at 3000 × g and then filtered through a 0.45 µm Millipore (Millipore, Bedford, MA) filter. The samples were pooled together and concentrated by means of “Centricon tubes” (Millipore, Bedford, MA) with a Mr cutoff of 3000 Da. Protein concentration of urines was determined using DC Protein Assay (Bio-Rad) by using bovine serum albumin (BSA) as standard. A total volume of 45 mL of sample was obtained, corresponding to 150 mg of protein content. This material was dialyzed against 25 mM sodium phosphate pH 7.0, then lyophilized and stored at -20 °C until use. Sample Treatment with Solid-Phase Ligand Library. The lyophilized urine was solubilized in 22 mL of 25 mM sodium phosphate buffer, pH 7.0. The solution was loaded onto a column (6.6 × 32 mm) of 1 mL of peptide library beads (“protein Equalizer technology” beads from Ciphergen Biosystems Inc. Fremont Ca, USA) at a flow rate of 0.24 mL/min. The column was then washed extensively with 25 mM phosphate buffer, pH 7.0 until the UV absorbance (214 nm) returned to baseline. Absorbed proteins were first eluted with TUC buffer (2.2 M thiourea, 7.7 M urea, 4.4% CHAPS) followed by 9 M urea, pH 3.8 (5% v/v acetic acid) using 3 column volumes (3 mL) each time. The collected elution fractions were immediately neutralized and frozen. SDS-PAGE Analysis. Urinary protein samples before and after treatment were analyzed by regular SDS-PAGE using a 16% polyacrylamide gel slab from Invitrogen (Carlsbad, Ca); experiments have been conducted under reduced and non reduced conditions. Protein staining was done by using both Coomassie colloidal blue and silver nitrate (Silverquest kit). 2-D-PAGE Analysis. For proper comparisons, the same protein concentration, i.e., 0.6 mg/mL, was used for all the samples investigated (starting material and the two obtained eluates). The desired volume of each sample (300 µL for the first elution, 820 µL for the second one and 22.5 µL for the starting material) was subjected to protein precipitation in a cold mixture of acetone and methanol (v/v ratio of 8:1), for removing lipids, salts and for regulating the concentration of protein samples, for 2 h at -20 °C and the solution was then centrifuged at 10 000 × g for 20 min. The pellet was solubilized in the “2-D sample buffer” (7 M urea, 2 M thiourea, 3% CHAPS, 40 mM Tris, 5 mM TBP and 10 mM acrylamide) and allowed to be alkylated at room temperature for 90 min. To stop the alkylation reaction, 10 mM DTT was added followed by 0.5% Ampholine and a trace amount of bromophenol blue to the solution. 7 cm long IPG strips (Bio-Rad), pH 3-10 were rehydrated with 150 µL of protein solution, for 4 h. Isoelectric

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The Hidden Human Urinary Proteome

focusing (IEF) was carried out with a Protean IEF Cell (BioRad), at an initial voltage of 1000 V for 15 h followed by applying a voltage exponential gradient up to 5000 V until each strip was electrophoresed for 25 kV × hours. For the second dimension, the IPGs strips were equilibrated for 26 min in a solution containing 6 M urea, 2% SDS, 20% glycerol, 375 mM Tris-HCl (pH 8.8) under gentle shaking. The IPG strips were then laid on an 8-18% acrylamide gradient SDS-PAGE with 0.5% agarose in the cathode buffer (192 mM glycine, 0.1% SDS and Tris to pH 8.3). The electrophoretic run was performed by setting a current of 5 mA/gel for 1 h, followed by 10 mA/gel for 1h and 20 mA/gel until the dye front reached the bottom of the gel. Gels were incubated in a fixing solution containing 40% ethanol and 10% acetic acid for 30 min followed by overnight staining in a ready-to-use Sypro Ruby solution. Destaining was performed in 10% methanol and 7% acetic acid for 1h, followed by a rinse of at least 3 h in pure water. The 2-DE gels were scanned with a Versa-Doc image system (BioRad), by fixing the acquisition time at 10 s; the relative gels images were evaluated using PDQuest software (Bio-Rad). After filtering the gel images to remove the background, spots were automatically detected, manually edited and then counted. SELDI Analysis. Protein solutions at appropriate concentration, i.e., 0.02 µg/µL, were deposited upon ProteinChip Array surfaces (Ciphergen Biosystems Inc.), using a bioprocessor. Two types of arrays were selected: CM10 (weak cation exchanger) and IMAC 30 (Immobilized Metal ions Affinity Capture) loaded with copper ions. Each array contained eight distinct spots over which the adsorption of protein could be performed. After applying the three samples all surfaces were dried and prepared for SELDI-TOF MS analysis by applying 1 µL of matrix solution composed of a saturated solution of sinapinic acid in 50% acetonitrile containing 0.5% trifluoroacetic acid. All arrays were then analyzed by using a Ciphergen PBSIIc ProteinChip Reader. The instrument was used in a positive ion mode, with an ion acceleration potential of 20 kV and a detector gain voltage of 2 kV. The mass range investigated was from 3 to 20 kDa. Laser intensity was set between 200 and 250 units according to the sample tested. The instrument was calibrated with “All-in-1 protein standard” mixture (Ciphergen Biosystems Inc.). Fourier Transform-Ion Cyclotron Resonance (FT-ICR) Mass Spectrometry (MS) Analysis. A 130-µg portion of each sample was precipitated by ethanol with glycogen as carrier and taken up in 8 M urea, 10 mM Tris pH 8.0. The proteins were reduced using DTT at 37 °C for 30 min and subsequently alkylated using iodoacetamide for 20 min in the dark at room temperature. The material was then diluted to 2 M urea using 100 mM ammonium bicarbonate and digested with 3 µg trypsin (Proteomics Grade, Sigma) at 37 °C overnight. The peptides were diluted 10-fold using 0.5% acetic acid and desalted using a StageTip with two C18-disks.35 Peptides were eluted with 50 µL 80% acetonitrile/0.5% acetic acid and concentrated to below 5 µL using a Speed Vac (Concentrator 5301, Eppendorf AG, Hamburg Germany). Volumes were adjusted to 5 µL by adding 1% trifluoroacetic acid and analyzed in a single run each on our LC-MS platform. The platform is composed of a LTQ-FT mass spectrometer (ThermoElectron, Bremen, Germany) and an Agilent 1100 binary nano pump (Palo Alto, CA). C18 material (ReproSil-Pur C18-AQ, 3 µm, Dr. Maisch GmBH, AmmerbuchEntringen, Germany) was packed into a spray emitter (75 µm ID, 8 µm opening, 70 mm length; New Objectives, USA) using an air-pressure pump (Proxeon Biosystems, Odense, Denmark) to prepare an analytical column with a self-assembled particle

Figure 1. Silver-stained Tris-Tricine SDS-PAGE of urine samples. Tracks: (a) starting material (control); (b) flow through; (c) first eluate (TUC); (d) second eluate (9 M urea, pH 3.8); (e) Mr markers.

frit.36 Mobile phase A consisted of water, 5% acetonitrile and 0.5% acetic acid and mobile phase B of acetonitrile and 0.5% acetic acid. The peptides were loaded at 700 nL/min flow rate. The gradient with 300 nL/min flow rate went for buffer B from 0% to 20% in 77 min and then in 5 min to 80%. The mass spectrometer acquired a MS at resolution 50.000 in the FTICR cell and eight MS/MS in the linear ion trap section per cycle. Peaks were picked using DTAsupercharge 0.62 (a kind gift from Matthias Mann, Odense, Denmark) with the parameters: precursor-peptide tolerance 0.2, SmartPicking enabled with max depth 8. The peak lists were searched against the IPI human database version 20050411 (http://www.ebi.ac.uk/IPI) using Mascot 2.0 with the parameters: monoisotopic masses, 0.2 Da on MS and 0.5 Da on MS/MS, quad-TOF parameter, carbamidomethylation for cysteine as fixed modification, oxidation on methionine and N-acetylation as variable modifications, two missed cleavage sites allowed. The significance threshold for the Mascot score was determined to be 18 by searching the data against the IPI database and a reversed IPI database to give a false positive rate of 5%. Proteins were accepted as identified if their total score was at least 36 resulting in an expected false positive rate of 0.25% or 1 in 400 (according to this last criterion, the original total of 470 identifications was reduced to only 383 entries, as listed in Table 1). Every peptide is assigned to a single protein. Proteins present in the list as different splice variants are matching to separate sets of peptides. Peptides shared between several proteins are only counted for the protein that has over all the most matching peptides. The presented protein list is hence the smallest set of proteins from the database that is needed to explain the presence of the identified peptides.

Results Figure 1 shows the SDS-PAGE profiles of the starting urine sample (a) and of the flow-through (b) after adsorption on the ligand library. Tracks (c) and (d), respectively, show the tracings of the eluate in TUC (thiourea/urea/CHAPS) and of the second eluate in 9 M urea at pH 3.8. Two phenomena are immediately apparent: (i) the drastically reduced levels of the most abundant proteins present in the control (notably albumin, Mr 67 kDa) and (ii) the presence of a continuum of bands covering the entire Mr interval from 10 up to >200 kDa in the two eluates after treatment with the ligand library. The dynamic range reduction effect is even more evident in the 2-D maps (Figure 2). The first eluate (TUC) exhibits many more spots in the entire pH interval (>300, as counted with the PDQuest) as compared with control urine (=100). InterestJournal of Proteome Research • Vol. 4, No. 6, 2005 1919

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Table 1. Summary of the Identified Proteins from Urine Samplesa

protein candidates

11 kDa protein 14 kDa phosphohistidine phosphatase 19 kDa protein 1-O-acylceramide synthase 21 kDa protein 38 kDa protein 4F2 cell-surface antigen heavy chain 62 kDa protein 6-phosphogluconolactonase 85 kDa protein 8D6 antigen Actin. cytoplasmic 1 Actin. cytoplasmic 2 Adenosylhomocysteinase ALB protein ALB protein ALB protein Alcohol dehydrogenase AlphA 3 type VI collAgen isoform 1 Alpha-1-acid glycoprotein 1 Alpha-1-acid glycoprotein 2 Alpha-1-antichymotrypsin Alpha-1-antitrypsin Alpha-1B-glycoprotein Alpha-2-antiplasmin Alpha-2-glycoprotein 1, zinc Alpha-2-HS-glycoprotein Alpha-actinin 4 Alpha-galactosidase A Alpha-N-acetylgalactosaminidase Alpha-N-acetylglucosaminidase AMBP protein Aminoacylase-1 Aminopeptidase N Angiopoietin-related protein 2 Angiotensinogen Annexin A1 Antibacterial protein FALL-39 Antileukoproteinase 1 Apolipoprotein Apolipoprotein A-I Apolipoprotein A-II Apolipoprotein A-IV Apolipoprotein D Apolipoprotein E Arylsulfatase A Aspartate aminotransferase, cytoplasmic Azurocidin Bactericidal/permeability-increasing protein-like 1 Basement membrane-specific heparan sulfate proteoglycan core protein Beta-2-glycoprotein I Beta-2-microglobulin Beta-galactosidase Betaine-homocysteine methyltransferase 2 Betaine--homocysteine Smethyltransferase Biotinidase BK65A6.2 Butyrophilin, subfamily 2, member A1, isoform 2 C219-reactive peptide Cadherin 11, type 2, isoform 1 preproprotein Cadherin-13 Cadherin-20 Cadherin-3 Calgranulin A Calgranulin B Carbonyl reductase [NADPH] 1 Carboxyl ester lipase

1920

accession no.

total no. combined of peptides mascot score identified

Mr/ Da

pI

10921 13995 19332 46913 21601 38940 58023 62984 27815 86207 29998 38505 42108 48124 48641 46442 73881 36761 345167 23725 23873 48668 46878 54809 54873 34451 40098 105245 49476 47047 82571 39886 46084 109711 57582 53406 22741 19517 15228 514737 30759 11282 45343 21547 36246 54613 46316

8.07 5.65 6.6 6.26 6.73 6.13 5.2 6.02 5.7 9.06 4.61 5.8 5.31 5.92 5.97 5.77 5.52 6.35 8.32 5.95 5.03 5.33 5.37 5.58 5.87 5.57 4.93 5.27 5.35 4.98 6.1 5.76 5.77 5.27 7.23 5.87 5.39 9.48 9.11 5.57 5.56 6.27 5.28 5.06 5.65 5.65 6.57

IPI00414741 IPI00299977 IPI00027388 IPI00301459 IPI00070778 IPI00515046 IPI00027493 IPI00166930 IPI00029997 IPI00179778 IPI00009850 IPI00550724 IPI00021440 IPI00012007 IPI00384697 IPI00216773 IPI00022434 IPI00220271 IPI00022200 IPI00022429 IPI00020091 IPI00556633 IPI00553177 IPI00022895 IPI00029863 IPI00166729 IPI00022431 IPI00013808 IPI00025869 IPI00414909 IPI00008787 IPI00022426 IPI00009268 IPI00221224 IPI00007800 IPI00032220 IPI00549413 IPI00292532 IPI00008580 IPI00029168 IPI00021841 IPI00021854 IPI00304273 IPI00006662 IPI00021842 IPI00329685 IPI00219029

1 2 1 3 1 1 1 4 4 4 2 5 7 2 7 1 58 3 2 5 1 2 13 19 1 17 14 1 6 2 10 54 5 7 1 2 2 1 2 1 8 2 2 38 11 2 3

40 95 57 126 56 46 50 195 168 149 118 193 405 69 397 57 2539 136 98 214 58 113 534 750 50 671 637 42 248 108 487 2800 255 359 48 70 88 42 64 68 400 80 77 2083 543 81 150

IPI00022246 IPI00296654

1 2

49 66

27325 6.35 49256 8.82

IPI00024284

26

1150

479248 6.06

IPI00298828 IPI00004656 IPI00441344 IPI00014363

1 3 1 1

91 109 47 39

39584 13820 76499 40841

IPI00004101

6

219

45398 6.4

IPI00218413 IPI00021302 IPI00019862

4 1 1

203 85 58

62006 5.5 91746 5.84 38228 5.91

IPI00419597 IPI00386476

3 10

203 445

56777 4.32 88381 4.77

IPI00024046 IPI00307612 IPI00216677 IPI00007047 IPI00027462 IPI00295386 IPI00099670

7 1 1 1 6 3 1

439 64 39 57 250 175 66

78694 89391 91884 10885 13291 30510 79959


x x

x x x x

x x x x x x x

10

x x

10,12 10, 12

x x x x x

x

x x

x x x x

x

x x x

x

x

x x

x

x x

x

x x

x x

x

10, 11, 18 9, 10, 11, 12, 18

x x x x

x

x x x x x x x x x x x

x

x x x x x x x

x

9, 10, 18 10, 12, 18 10, 18 18 9, 10, 18

x

10 9 9, 10, 18 12 18

x x x

x

x

x

x

10, 18 x

10, 11, 12, 18 9, 18 10 10

x x x

8.34 6.06 6.1 5.61

4.8 4.54 4.62 6.51 5.71 8.55 5.13

refs where the candidate was also observed

exp. 1 exp. 1 exp. 1 exp 2 exp 2 exp 2 2nd 1st 2nd urine 1st urine tot elution elution tot elution elution

x

x

x x

x

9, 10, 18 9, 18 9, 10, 18

x x x x

x

x

x x x

x

x x

x x

x

x

x x x

x

x

x x x

x

11 x 10 10, 11, 18

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The Hidden Human Urinary Proteome Table 1. (Continued)

protein candidates

Cartilage intermediate layer protein-like protein CLIP-2 Cartilage oligomeric matrix protein Cathepsin B Cathepsin D Cathepsin G Cathepsin H Cathepsin L Cathepsin Z CD44 antigen CD59 glycoprotein Ceruloplasmin Chloride intracellular channel protein 1 Cholesteryl ester transfer protein Clusterin Coactosin-like protein Collagen alpha 1(VI) chain Collagen alpha 2(IV) chain Collectrin Complement C3 Complement C4 Complement factor H-related protein 1 Complement factor I Creatine kinase, B chain Cystatin C Cystatin S Cytosolic nonspecific dipeptidase Deoxyribonuclease-1 Dermatopontin Dipeptidyl-peptidase I Dipeptidyl-peptidase II DTTR431 E-cadherin EGF-containing fibulin-like extracellular matrix protein 2 Endothelial protein C receptor Ephrin type-B receptor 6 EWI2 Extracellular superoxide dismutase [Cu-Zn] Fatty acid-binding protein, epidermal Fc fragment of IgG binding protein Fc of IgG, low affinity IIIa, receptor for Ferritin light chain Fibrillin 1 Fibulin-5 FLJ90165 protein Fructose-1,6-bisphosphatase Fructose-bisphosphate aldolase A Fructose-bisphosphate aldolase B G7c protein Galectin-3 binding protein Ganglioside GM2 activator Gelsolin Glutamine synthetase Glutaminyl-peptide cyclotransferase Glutamyl aminopeptidase Glutathione S-transferase A2 Glutathione S-transferase P Glyceraldehyde-3-phosphate dehydrogenase, liver Golgi apparatus protein 1 Golgi phosphoprotein 2 Growth-arrest-specific protein 1 Haptoglobin-related protein HBB protein Heat-shock protein beta-1 Hemicentin Hemopexin Hepatocellular carcinoma associated protein TB6 High mobility group protein 4 Histidine-rich glycoprotein Histone H2B.q Histone H4/o

accession no.


Mr/ Da

pI


exp. 1 exp. 1 exp. 1 exp 2 exp 2 exp 2 2nd 2nd urine 1st 1st urine tot elution elution tot elution elution

IPI00216780

3

119

128478 8.63

x

IPI00028030 IPI00295741 IPI00011229 IPI00028064 IPI00297487 IPI00012887 IPI00002745 IPI00419219 IPI00011302 IPI00017601 IPI00010896 IPI00006173 IPI00291262 IPI00017704 IPI00291136 IPI00306322 IPI00010191 IPI00164623 IPI00032258 IPI00011264 IPI00291867 IPI00022977 IPI00032293 IPI00032294 IPI00177728 IPI00031065 IPI00292130 IPI00022810 IPI00296141 IPI00243602 IPI00000513 IPI00296058

1 2 2 1 2 3 6 2 8 7 1 1 12 3 11 1 1 8 5 2 2 1 3 1 2 1 2 6 2 1 5 3

75 68 110 47 80 136 273 75 377 281 47 42 454 98 416 69 49 427 258 114 107 40 121 39 86 39 122 318 66 38 237 130

85402 38752 45037 29161 37980 37996 34530 53879 14795 122983 27117 55134 53031 15918 109621 168628 25447 188585 194247 38777 68072 42902 16017 16489 53187 31642 24559 52607 54749 33168 91342 51753

4.34 5.88 6.1 11.19 8.38 5.32 6.7 5.18 6.02 5.44 5.09 5.7 5.89 5.55 5.25 8.89 5.43 6.02 6.66 7.75 7.72 5.34 9 4.95 5.66 4.71 4.7 6.53 5.91 6.15 4.61 4.79

x

IPI00009276 IPI00005222 IPI00056478 IPI00027827

7 2 2 21

211 113 100 908

26997 110800 65621 26321

6.7 6.19 8.23 6.13

IPI00007797 IPI00242956 IPI00218834 IPI00375676 IPI00328113 IPI00294615 IPI00440764 IPI00073772 IPI00549682 IPI00218407 IPI00008893 IPI00023673 IPI00018236 IPI00026314 IPI00010130 IPI00003919 IPI00014375 IPI00552774 IPI00219757 IPI00219018

3 5 4 1 5 4 1 4 2 13 1 10 5 3 2 6 2 2 1 1

136 244 181 69 257 201 46 258 115 652 48 601 284 152 60 238 102 61 103 44

15366 596522 33126 28684 332682 52485 51076 37059 52762 39830 58144 66202 21265 86043 42665 40965 109689 25531 23438 36070

6.82 5.56 8.2 5.51 4.41 4.58 5.66 6.61 8.39 8.06 5.56 5.13 5.17 5.9 6.43 6.12 5.34 8.54 5.44 8.58

IPI00414717 IPI00171411 IPI00008832 IPI00296170 IPI00218816 IPI00025512 IPI00045512 IPI00022488 IPI00293898

3 3 3 1 2 1 2 4 7

92 101 178 44 129 66 101 137 237

141009 46416 36895 44076 16102 22826 623613 52385 84429

6.53 4.91 5.35 6.66 6.81 5.98 6.05 6.81 5.59

IPI00217477 IPI00022371 IPI00003935 IPI00453473

1 1 3 2

50 48 105 80

23005 60510 13781 11360

8.5 5.71 10.32 11.36

x

x

x x

x

x x x

10

x x x x

x

x 9, 18 9, 10, 18 10, 18

x x x x

x

x x x x x x x x x x

10, 11, 18

x

18 9, 18

x x x

11 x

x

10, 11 x

x

x

x x x x x

9 10

x x x

x x

10, 11, 12, 18

x

10, 18 x

x

x x

x

x

x

x

x

x x

x

10 10 10

x x

x x

x x

x x

x x x

x x

x x x x x x x x

x 9 9, 10 10, 12, 18

x x x x x x

9, 10, 11, 18 10

x x

x x

x x x x x x x

x

10 10 9, 10, 11, 12, 18

x x x x

x

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Table 1. (Continued)

protein candidates

HLA class I histocompatibility antigen, Cw-3 alpha chain HP protein HSD-33 HYAL1 protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein DKFZp451K1918 Hypothetical protein DKFZp686I04196 Hypothetical protein DKFZp686I15212 Hypothetical protein FLJ14847 Hypothetical protein FLJ16045 Hypothetical protein FLJ22662 Hypothetical protein FLJ42063 Hypothetical protein FLJ42598 Hypothetical protein LOC196463 Ig alpha-1 chain C region Ig alpha-1 chain C region Ig heavy chain V-III region BRO Ig heavy chain V-III region TIL Ig kappa chain C region Ig kappa chain V-III region B6 Ig kappa chain V-III region HAH Ig kappa chain V-III region SIE Ig kappa chain V-IV region Len Ig lambda chain C regions Ig lambda chain V-III region SH Ig lambda chain V-IV region Hil IGHA1 protein IGHG3 protein IGHM protein IGKV1-5 protein IGKV1-5 protein IGLC1 protein IGLV2-14 protein ImmunoglobulIn J chaIn Insulin-like growth factor binding protein 7 Intimal thickness-related receptor Intrinsic factor-B12 receptor ISLR Kallikrein 1 Keratin 13 isoform b Keratin 9 Keratin, type I cytoskeletal 10 Keratin, type II cytoskeletal 1 Keratin, type II cytoskeletal 6A Keratin, type II cytoskeletal 6B Lactotransferrin Lambda-crystallin Latent transforming growth factorbeta-binding protein 2 Lectin-like oxidized LDL receptor Leucine-rich alpha-2-glycoprotein Leukocyte antigen CD84 Leukocyte-associated Ig-Like receptor 1 isoform b Limitrin Lithostathine 1 beta L-lactate dehydrogenase A chain L-lactate dehydrogenase B chain Low-density lipoprotein receptorrelated protein 2 L-selectin L-xylulose reductase Lymphatic endothelium-specific hyaluronan receptor LYVE-1 Lysosomal acid phosphatase Lysosomal alpha-glucosidase

1922

accession no.

total no. combined of mascot peptides score identified

Mr/ Da

pI

41234

5.97

x

31647 8.48 46821 8 45729 7.05 26503 8.24 26103 6.14 25122 8.62 52148 6.56 51939 7.55 66998 6.53 25350 7.59 39876 5.2 46716 7.66 58123 8.39 55861 5.27 95117 5.62 63374 9.11 38147 4.93 30278 5.94 65886 6.53 54190 5.78 56741 7.58 13332 6.44 12462 9.23 25800 6.3 11742 9.34 14178 7.75 11882 8.7 12746 7.92 65755 11.36 11500 6.02 11624 6.04 53858 6.74 58374 7.95 53317 7.5 26034 5.74 26503 6.3 25119 5.93 24722 6.81 18543 4.62 30138 8.25

x x x x

IPI00472612

1

46

IPI00431645 IPI00418446 IPI00168847 IPI00550731 IPI00549747 IPI00447449 IPI00441196 IPI00439447 IPI00430856 IPI00386158 IPI00297160 IPI00399007 IPI00418153 IPI00043992 IPI00419215 IPI00016255 IPI00171871 IPI00446210 IPI00169285 IPI00550584 IPI00549485 IPI00382480 IPI00382478 IPI00549330 IPI00387113 IPI00030205 IPI00387115 IPI00387120 IPI00551005 IPI00382436 IPI00382440 IPI00430842 IPI00472345 IPI00472610 IPI00430820 IPI00419424 IPI00154742 IPI00552195 IPI00178926 IPI00016915

2 4 5 8 2 1 3 3 3 7 1 6 4 2 2 1 1 2 2 8 3 1 1 1 2 2 3 3 11 2 1 12 2 9 4 7 2 1 6 18

90 125 241 426 68 41 128 140 99 331 43 180 155 112 77 51 53 100 117 348 178 48 59 49 75 74 192 167 439 101 83 421 61 359 155 359 63 49 233 899

IPI00305011 IPI00160130 IPI00023648 IPI00304808 IPI00171196 IPI00019359 IPI00009865 IPI00220327 IPI00300725 IPI00293665 IPI00298860 IPI00385216 IPI00292150

2 15 3 5 5 10 1 11 12 2 5 1 5

105 768 107 315 238 469 66 601 613 109 179 52 195

50104 407465 46596 29498 46179 62255 59711 66018 60162 60116 80170 35909 204059

7.01 5.12 5 4.62 4.91 5.14 5.13 5.14 5.14 8.14 8.56 5.81 5.08

IPI00001759 IPI00022417 IPI00022039 IPI00028015

1 10 2 2

47 556 80 117

31453 38382 37133 30106

6.94 6.1 8.52 5.86

IPI00550854 IPI00009197 IPI00217966 IPI00219217 IPI00024292

2 1 2 5 16

120 48 76 229 767

49500 19052 36819 36769 540349

6.75 5.67 8.46 5.72 4.9

IPI00218795 IPI00448095 IPI00290856

1 1 1

54 59 45

45128 26182 35729

6.2 8.33 8.76

IPI00003807 IPI00293088

5 14

202 550

48685 106649

6.14 5.62


exp 2 exp. 1 exp. 1 exp. 1 exp 2 exp 2 2nd 1st 2nd urine 1st urine tot elution elution tot elution elution

x


10

x x x x x x x x x x x x x x x x x

x x x

x x

x x

x x

x x x x

x x x

x

10, 18 10, 18 18 9, 10, 12, 18 10, 18

x 10, 18 9, 10, 18

x x x

x

x x x x

x x

x

x

x x x x

x

x

x

x

x

x x

x

x x x x x x

x x x

x

10 10, 18

x x x x x x

x x x x

18 10 9 9 9 9 9 9

x x

x x

x

x

x x

10, 11

x

9, 18

x x x

x

x x x

x

x

9, 10 x 12

x x x

x

x x

x x

10 10

research articles


protein candidates

Lysosomal Pro-X carboxypeptidase Lysozyme C Major prion protein Malate dehydrogenase, cytoplasmic Mammalian ependymin related protein 1 Mannosyl-oligosaccharide 1,2-alphamannosidase IA Melanoma chondroitin sulfate proteoglycan Microsomal dipeptidase Moesin Monocyte differentiation antigen CD14 Multimerin 2 Muscle-cadherin Myosin-reactive immunoglobulin kappa chain variable region N-acetylglucosamine-6-sulfatase N-acetyllactosaminide beta-1,3-Nacetylglucosaminyltransferase Napsin A Natural killer cell-specific antigen KLIP1 Neprilysin Neural cell adhesion molecule Neural-cadherin Neuronal growth regulator 1 Neuroserpin Neutrophil defensin 1 NG,NG-dimethylarginine dimethylaminohydrolase 2 Nicotinate-nucleotide pyrophosphorylase Nidogen Nonsecretory ribonuclease NOV protein homolog Novel protein N-sulphoglucosamine sulphohydrolase Nuclear transport factor 2 Nuclease sensitive element binding protein 1 Nucleobindin 1 Osteoclast-associated receptor hOSCAR-M3 Pancreatic alpha-amylase Pancreatic secretory trypsin inhibitor PCDH12 protein Pepsin A Peptidoglycan recognition protein Peptidyl-prolyl cis-trans isomerase A Peripherin Peroxiredoxin 1 Peroxiredoxin 6 Phosphatidylcholine-sterol acyltransferase Phosphatidylethanolamine-binding protein Phosphoglycerate kinase 1 Pigment epithelium-derived factor Plasma glutathione peroxidase Plasma protease C1 inhibitor Plasma retinol-binding protein Plasma serine protease inhibitor Plasminogen Plasminogen/activator kringle Poly Polymeric-immunoglobulin receptor PREDICTED: hypothetical protein BC004360 PREDICTED: similar to Adrenoleukodystrophy protein (ALDP) PREDICTED: similar to PI-3-kinaserelated kinase SMG-1 isoform 1 PREDICTED: similar to POTE2A PREDICTED: similar to Triosephosphate isomerase (TIM)

accession no.


Mr/ Da

pI

56277 16982 27871 36500 38572 73150

6.76 9.38 9.75 6.89 6.33 6.04

IPI00001593 IPI00019038 IPI00022284 IPI00291005 IPI00259102 IPI00291641

2 5 1 3 3 2

62 163 50 139 149 79

IPI00019157

8

396

251027 5.2


1 1 12 2 3 2

56 51 582 92 153 115

46091 67761 40678 105036 89261 11868

IPI00012102 IPI00009997

3 2

174 102

x


1 1 3 2 1 1 1 2 6

71 38 131 62 65 44 49 81 266

IPI00300086

2

80


7 2 1 9 1

311 92 41 461 81

IPI00009901 IPI00031812

6 1

187 44

IPI00295542 IPI00107731

3 2

171 72

IPI00025476 IPI00020687 IPI00009851 IPI00019641 IPI00021085 IPI00419585 IPI00013164 IPI00000874 IPI00220301 IPI00022331

6 2 2 2 9 4 1 2 1 1

224 67 86 76 529 158 43 93 81 40

IPI00219446

4

117

21027 7.56

x

IPI00169383 IPI00006114 IPI00026199 IPI00291866 IPI00022420 IPI00007221 IPI00019580 IPI00386265 IPI00016610 IPI00004573 IPI00042295

4 4 4 8 8 16 22 1 1 4 2

245 194 131 353 260 653 903 40 38 199 95

44854 46484 25774 55347 23337 45787 93247 10140 37987 84459 39419

x x x x x x x

IPI00397198

1

39

142369 10.33

IPI00399173

1

50

189231 8.66

IPI00248359 IPI00383071

1 4

46 224

119319 5.93 27211 8.21

x x

x x

x x x

x

x

x

5.75 6.09 5.84 5.5 4.81 8.74

x

x

x

x x

6.15 5.47 5.54 5.54 4.64 6.1 4.84 6.54 5.66

5.16 9.1 8.12 6.42 6.46

x x x x x

11 18

x

18

x x x x x x

x

x

10

x x

x

x x

18

x

14640 5.1 35903 9.87

x

x

53818 5.12 27897 8.88

x x

8.3 5.97 8.2 6.09 5.45 5.42 7.04 6.68 6.66 5.59 6.19

9, 10, 12, 18

x

x x

6.6 7.54 5.11 4.16 8.92 7.82 5.43 8.27 6.02 9.13

x x

x x

x

58354 8843 129837 42350 22116 18098 53960 22324 25002 49888

x

x

31138 5.81 139254 18855 41473 49988 57173

9

x

x

62840 8.06 47545 6.77 45700 38417 86013 137652 100246 39379 46397 10536 29911



10 x

x

x

x x

9, 10, 11, 12, 18 x

x x

x x

x

x x

x x x

9 x x x x x x

10, 18 9, 18 9 9, 10, 12 x

x x x

x x

10, 18 x

x x x x

x


research articles

Castagna et al.


protein candidates

Probable endonuclease KIAA0830 Pro-epidermal growth factor Profilin-1 Prolactin-inducible protein Prostaglandin D2 synthase 21kDa Prostaglandin-H2 D-isomerase Prostate specific antigen Prostate stem cell antigen Prostatic acid phosphatase Proteasome subunit beta type 6 Protein FAM3C Protein tyrosine Phosphatase, receptor type, sigma isoform 3 Protein UNQ6350/PRO21055 Prothrombin Prothymosin alpha Prothymosin alpha Puromycin-sensitive aminopeptidase Pyruvate kinase 3 isoform 2 Quiescin Q6 Resistin Retinal dehydrogenase 1 RGTR430 Ribonuclease 4 Ribonuclease pancreatic RLLV422 S100 calcium-binding protein A7 Secreted and transmembrane protein 1 Serotransferrin SERPINC1 protein Serum amyloid P-component SH3 domain binding glutamic acid-rich protein like 3 Sialidase 1 Similar to CG3714 gene product Small breast epithelial mucin Sorbitol dehydrogenase Sortilin Splice Isoform 1 of Acyl-protein thioesterase 1 Splice Isoform 1 of Amiloridesensitive amine oxidase [coppercontaining] Splice Isoform 1 of Anthrax toxin receptor 1 Splice Isoform 1 of Brevican core protein Splice Isoform 1 of Cadherin-16 Splice Isoform 1 of Cadherin-6 Splice Isoform 1 of CCG1-interacting factor B Splice Isoform 1 of Complement decay-accelerating factor Splice Isoform 1 of Complement factor B Splice Isoform 1 of Complement factor H Splice Isoform 1 of EGF-containing fibulin-like extracellular matrix protein 1 Splice Isoform 1 of Extracellular sulfatase Sulf-2 Splice Isoform 1 of Fibronectin Splice Isoform 1 of Folate receptor alpha Splice Isoform 1 of Gammaglutamyltranspeptidase 1 Splice Isoform 1 of ICOS ligand Splice Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H4 Splice Isoform 1 of Latrophilin 1 Splice Isoform 1 of Leukocyte immunoglobulin-like receptor subfamily B member 4 Splice Isoform 1 of Mannan-binding lectin serine protease 2 Splice Isoform 1 of Melanotransferrin Splice Isoform 1 of N-acetylmuramoyl-Lalanine amidase Splice Isoform 1 of Nucleophosmin

1924

accession no.


Mr/ Da

IPI00001952 IPI00000073 IPI00216691 IPI00022974 IPI00513767 IPI00013179 IPI00010858 IPI00013446 IPI00396434 IPI00000811 IPI00021923 IPI00293275

3 67 1 6 5 8 11 2 8 1 1 1

145 3155 58 266 235 344 406 114 334 46 60 43

55723 137565 15085 16847 23050 21243 29293 13474 44880 25570 24950 169480

5.55 5.56 8.47 8.26 9.92 7.66 7.61 5.07 5.83 4.8 5.7 8.52

IPI00216914 IPI00019568 IPI00412977 IPI00337654 IPI00026216 IPI00220644 IPI00003590 IPI00006988 IPI00218914 IPI00066856 IPI00029699 IPI00014048 IPI00152018 IPI00219806 IPI00170635 IPI00022463 IPI00165421 IPI00022391 IPI00514669

16 34 3 4 1 5 8 5 1 1 2 5 1 1 5 15 2 2 1

710 1579 88 282 42 279 354 247 49 65 63 231 47 45 303 707 81 73 46

22034 71475 11952 8156 103895 58538 83324 12096 55323 13791 17286 18089 36576 11433 27307 79280 29473 25485 9432

4.9 5.64 3.65 3.71 5.49 7.95 9.13 6.52 6.29 5.69 9.3 9.1 5.68 6.26 7 5.69 9.03 7.09 9.42


6 1 1 2 1 1

236 45 39 89 58 57

45952 50002 9091 38768 93011 24996

5.59 5.36 4.41 8.25 5.46 6.29

IPI00020982

1

40

85709 6.6

IPI00030431

1

45

63433 7.54

IPI00456623 IPI00025240 IPI00024035 IPI00063827

1 3 7 1

41 126 312 55

IPI00216550

2

IPI00019591 IPI00029739 IPI00029658

x

x

x

x x

x x x x

x x

x x x x

x

x x x x x

x x x

x x

x x

x

x

x

x x

x x

x x

9, 18 9 9 9, 10, 18 10, 18 18 9, 10, 12, 18 10. 18

x

x x x x x

x x x

9, 18

x x

x x x x x x x

x x x

18 x x x x

10 9, 10, 11, 12, 18 18 10, 11

x x x x x x x x x

4.59 4.82 4.77 5.94

x

58

49713 7.79

x

1 3 12

49 92 497

86847 6.67 143710 6.28 56885 4.95

x

IPI00297252

1

50

101759 9.3

x

IPI00022418 IPI00441498 IPI00018901

2 3 3

77 154 135

266034 6.45 30712 7.57 61686 6.65

IPI00219131 IPI00294193

3 7

202 250

33841 5.15 103522 6.51

IPI00183445 IPI00289926

3 1

126 64

164609 6.13 49609 6.28

IPI00294713

9

297

77176 5.43

x

IPI00029275 IPI00163207

3 1

155 64

81787 5.66 62748 7.25

x

IPI00027499

2

83

32726 4.64


100538 90380 88539 22446


exp. 1 exp. 1 exp. 1 exp 2 exp 2 exp 2 2nd 2nd urine 1st 1st urine tot elution elution tot elution elution pI

x x

x

x x

x x

x x x

18 18

x

9, 10, 18

x

12

x

9, 10, 18

x

x

x x

x

x x

x

x x x

research articles


protein candidates

Splice Isoform 1 of Pappalysin-2 Splice Isoform 1 of Phosphoserine aminotransferase Splice Isoform 1 of Programmed cell death 1 ligand 2 Splice Isoform 1 of Protocadherin alpha 6 Splice Isoform 1 of Protocadherin gamma C3 Splice Isoform 1 of Roundabout homolog 4 Splice Isoform 1 of Tripeptidyl-peptidase I Splice Isoform 1 of Tubulointerstitial nephritis antigen-like Splice Isoform 1 of Vitamin K-dependent protein Z Splice Isoform 1 of V-set and immunoglobulin domain containing protein 4 Splice Isoform 1 of WAP four-disulfide core domain protein 2 Splice Isoform 2 of Granulins Splice Isoform 2 of Inter-alpha-trypsin inhibitor heavy chain H4 Splice Isoform 2 of Vitamin K-dependent protein Z Splice Isoform 2 of WAP four-disulfide core domain protein 2 Splice Isoform 2A of Desmocollin-2 Splice Isoform 2C2A of Collagen alpha 2(VI) chain Splice Isoform A of Ketohexokinase Splice Isoform A of Lamin A/C Splice Isoform A of Osteopontin Splice Isoform Alpha of Stromal cellderived factor 1 Splice Isoform Alpha-E of Fibrinogen alpha/alpha-E chain Splice Isoform APP770 of Amyloid beta A4 protein Splice Isoform B of Fibulin-1 Splice Isoform Beta of Pancreatic secretory granule membrane major glycoprotein GP2 Splice Isoform Beta of Poliovirus receptor Splice Isoform Delta of Poliovirus receptor related protein 2 Splice Isoform H17 of Myeloperoxidase Splice Isoform LMW of Kininogen Splice Isoform Long of Tyrosine-protein kinase receptor UFO Splice Isoform Mitochondrial of Glycine amidinotransferase, mitochondrial Splice Isoform Sap-mu-0 of Proactivator polypeptide Squamous cell carcinoma antigen 1 Tetranectin Thrombomodulin Thrombospondin 1 Thyrotropin-releasing hormone degrading ectoenzyme Thyroxine-binding globulin TiTin isoform novex-1 Transthyretin Trefoil factor 2 Trefoil factor 3 Tropomyosin 4 TSLC1-like 2 Tumor endothelial marker 1 Tumor necrosis factor receptor superfamily member 14 Urokinase-type plasminogen activator Uromodulin Uteroglobin V2-17 protein Vacuolar ATP synthase subunit S1 Vasorin Vesicular integral-membrane protein VIP36

accession no.


Mr/ Da

pI

IPI00013569 IPI00001734

2 1

85 53

203458 5.26 40796 7.56

IPI00414542

1

53

31279 8.18

IPI00009042

1

45

103336 4.93

x

IPI00001872

1

49

101301 5.07

x

IPI00103871 IPI00298237 IPI00005563

6 6 2

338 327 71

108417 6.18 61704 5.97 53721 6.54

IPI00027843

5

160

46026 5.64

x

IPI00027038

1

52

44529 5.93

x

IPI00291488

5

219

13953 4.69

x

IPI00182138 IPI00218192

4 5

222 232

50607 6.43 101521 6.51

IPI00216065

2

76

48391 5.64

IPI00103636

3

142

8571 4.69

IPI00025846 IPI00220613

3 1

107 50

101324 5.19 98506 5.78

IPI00029488 IPI00021405 IPI00021000 IPI00216304

1 1 6 1

67 49 320 52

33109 74380 35572 10382

IPI00021885

7

239

95656 6.27

IPI00006608

7

248

87914 4.73

IPI00218803 IPI00299429

2 1

149 51

81126 5.11 43086 4.98

IPI00219425 IPI00022661

6 1

181 86

40442 6.07 58162 5.31

IPI00007244 IPI00215894 IPI00296992

9 54 5

356 2311 262

84784 9.19 48936 6.34 98572 5.19

IPI00032103

1

62

48938 8.26

IPI00012503

2

125

59899 5.06


7 5 1 3 1

329 252 67 131 46

44594 22951 63083 133291 117439

6.4 5.52 4.78 4.17 6.5


2 2 1 3 2 1 1 33 1

87 58 62 133 77 53 53 1268 74

46637 3025342 15991 15130 14870 28619 43215 82803 31683

5.87 6.35 5.43 5.51 5.66 4.67 5.92 5.18 6.93

IPI00296180 IPI00013945 IPI00006705 IPI00552385 IPI00020430 IPI00395488 IPI00009950

14 40 4 1 1 23 9

582 1789 173 52 72 1050 290

49919 72451 10215 10513 52164 72751 40545

8.78 5.05 4.99 4.46 5.73 7.16 6.46

5.6 6.57 4.37 9.92



x

10 x

x

x x

x

x x

x x x

9 x

18

x

x

10, 18 x

9, 18

x

18 x x

9 x x

x

x

x

x

x x

x

x

x

18

x

18

x

x

x

x x

x

x x x x x

x x x

x x

x x

x

9, 10, 12, 18 18

x x x x

x

9, 10

x x

x x x

10, 18

x x

9, 10, 12, 18 10

x x x x x x

x

x x x x x x

10

x

x

x x

x x x x x

x

x

x x

9, 10, 18 18

x x x x


research articles

Castagna et al.


protein candidates

accession no.

Vitamin D-binding protein Vitamin K-dependent protein C Vitamin K-dependent protein S Vitronectin WNT1 inducible signaling pathway protein 2 WUGSC:DJ515N1.2 protein


total no. combined of mascot peptides score identified

1 1 2 18 2 2

40 66 134 786 77 84

Mr/ Da

pI

54499 53406 77127 55069 28460 28686

5.33 5.85 5.48 5.55 6.11 4.92

refs where the exp. 1 exp. 1 exp. 1 exp 2 exp 2 exp 2 candidate was also 2nd 1st 2nd urine 1st urine tot elution elution tot elution elution observed

x x x

x

x x x

x

x x

11, 18 9 9 x

x

a

The IPI database accession number, the total number of peptides identified and the MASCOT score are listed. Theoretical Mr and pI, as resulted from Compute pI/Mw tool of Expasy (http:// www.expasy.org/tools/pi_tool.html) are also indicated. Additional information about the presence of the identified proteins in the total urine or in the first or second eluate, as well as in the listed references, are shown.

Figure 2. Two-dimensional maps of urines. A: control; B: first eluate (TUC); C: second eluate (9 M urea, pH 3.8). Staining with Sypro Ruby. An equal load of total protein was applied for all samples analyzed (90 µg total protein/gel).

ingly, the second eluate, although displaying a significantly lower number of spots (=120), shows only a limited redundancy (common spots) with the TUC eluate, most of desorbed proteins being specific of the second elution step. Also SELDI MS profiling analysis related to the same protein fractions (Figure 3) (as limited to two M/z windows, one covering the 2500-7500 range, upper panel and the other over the 750020000 M/z interval, lower panel) confirms the above data, suggesting a total of at least 3 times as many signals as compared with the control. Actually, the number of peaks from control urine was 84 as compared to 135 and 146 peaks counted for respectively TUC and acidic urea eluate. Within the explored interval (2500-20000 Da), the total number of unique nonredundant peaks revealed by this analysis was 224 or almost 3 times larger than that of the control. It is here interesting to note that the peak count was limited only to proteins that interact with the IMAC-Cu2+ surface. These three samples were then subjected to FT-ICR analysis as described above. The data are listed in Table 1: control urines revealed a total of 134 unique gene products. On the contrary, the TUC eluate allowed identification of 317 unique protein species and the second eluate of an additional 95 1926


species. It is of interest to calculate which polypeptide chains are unique to each sample, and thus to obtain the degree of redundancy. This is shown schematically in Figure 4, that gives the degree of overlap of the various protein species in the different fractions (this could also be derived from Table 1, where a plus sign gives the presence of a given protein in each of the three fractions). By eliminating the redundancies and summing up all the species detected, we arrive at a total count of 383 unique protein species identified in urines. We have additionally represented the above data in the bar graph of Figure 5, which gives the increment in species obtained in the sum of the two eluates, as compared with the control, while simultaneously expressing their Mr distribution. The latter is a skewed distribution with a peak in the Mr 30 to 50 kDa range, as expected due to the filtering properties of the glomeruli. The significance of these results will be highlighted below. The identified proteins, listed in Table 1, were also classified according to their different molecular functions, and the results of such analysis are summarized in Figure 6. Most of the proteins seem to have binding properties, followed by catalytic and signal transducer, respectively.


research articles

Figure 3. Narrow extract of SELDI MS data obtained using an IMAC-Cu2+ ProteinChip Array covering two molecular mass ranges (2500-7500 Da, upper panel and 7500-20 000 Da, lower panel). “a” represents the initial crude urine proteins with affinity for IMACcopper ions surface; “b” represents the first elution using TUC buffer and “c” the second elution using 9 M urea, pH 3.8. (For more details see the Material and Methods section).

to add the 38 different urinary proteins listed in the Danish Centre for Human Genome Research (http://biobase.dk/cgibin/celis).

Figure 4. Diagram of overlap among the proteins identified in total urines (134 unique proteins), first (317 species) and second (95 pecies) eluates.

Discussion Up to the present study, the total detection of proteins in human urines could be summarized as follows: (a) 124 gene products in Spahr et al.18 (results obtained via liquid chromatography-tandem mass spectrometry of peptide fragments); (b) 103 species in Pang et al.9 (sum of data from 2-D maps, 1-D and 2-D liquid chromatography); (c) 150 unique protein annotations in Pieper et al.10 (as obtained via extensive sample pre-fractionation and 2-D map analysis); (d) 113 different proteins in Oh et al.11 (from 2-D gel mapping followed by peptide mass fingerprinting); (e) 47 unique proteins in Thongboonkerd et al.12 (total sum of those polypeptides found in two fractions, acetone-precipitated and ultracentrifugation collected); (f) 48 nonredundant proteins in Smith et al.16 (but only 22 reported in their Table 1; as obtained by adsorbing urinary proteins into a C18 resin and eluting at progressively higher levels of acetonitrile, from 30 to 70%). To those, one might want

It is intriguing to see what fraction of these proteins is in common among all the reported databases just quoted. The total sum of unique proteins reported in refs 9-12,18 is 537. When checked against our Table 1, 132 were found to be in common, thus leaving 251 unique species identified in the present report. This leaves out some 269 entries as available only in refs 9-12,18. Thus, by summing up our data with those already existing in the literature, it would appear that in urine, we can identify ca. 800 gene products, definitely a huge improvement from the early papers published in the 1980s, when just about a dozen proteins could be identified in 2-D maps, mostly of serum origin, and essentially only via immunoblots.4 What fraction of the total proteins possibly present in human urines would these 800 proteins represent is difficult to determine at present. Some considerations can be here expressed. First of all, it was surprising for us to see that 30 proteins, found in the control urines (and representing ca. 7% of the total proteins detected), were missing in the two bead eluates, since previous results gave such a loss at a 2-3%.31 The capturing mechanism, given the short length of the bait, should be mostly via classical physicochemical interactions among complementary polypeptide chains, such as ion exchange, hydrogen bonds, van der Waals and hydrophobic interactions.37 These interactions are present concomitantly under a combinatorial configuration inducing thus a panel of interactions ranging from rather weak dockings to very strong associations. Journal of Proteome Research • Vol. 4, No. 6, 2005 1927

research articles

Castagna et al.

Figure 5. Number of species detected in the two combined eluates (amaranth bars) as compared with control (turquoise bars) urines, as a function of their respective Mr values in the 5 kDa >200 kDa range.

Figure 6. Classification of all the proteins listed in Table 1 according to their molecular function, obtained with the program FatiGO(www.fatigo.org): note that single proteins may belong to more categories, which explains the total sum being substantially larger than 100%.

Weak interactions could potentially lead to instability in capturing some proteins from the sample or their release upon the wash step before the elution. Conversely, very strong interactions may induce difficulties in the desorption of captured species. Given the observed elution behavior, it is out of doubt that a number of interactions appear to be rather strong. TUC alonespretty drastic denaturing solutionsdoes not release all the species adsorbed. A second elution in 9 M urea under acidic conditions is necessary to release a substantial number of additional proteins, not present in the first eluate. Other desorption agents have been investigated, such as 6 M guanidinium hydrochloride (GuHCl), a well-known chaotrope able to disrupt all non covalent bonds; however, this had been abandoned in the present study because it was not compatible with the following analytical methods. The removal of GuHCl from the eluate resulted in a severe loss of proteins. Thus, we focused on two-step elution involving TUC and acidic urea solution, since both are more compatible with 2-D map analysis. The interaction between urinary proteins and ligands from the library is not only dependent on affinity constants; large proteins may be adsorbed on beads by a multipoint possible interaction resulting in a stronger apparent association constant. In this situation, elution could become very difficult explaining that a limited number of proteins may still be adsorbed on beads and hence non detected by our analytical approaches. Another possible scenario that could represent the 1928


limit of this process is the absence among the numerous structures of ligands having an affinity for some proteins; in this specific circumstance, few proteins may escape to the concentration effect. This could also be the case when the dissociation constant has values above the initial concentration of the protein to be captured. Although these two situations could be real, they have a low probability due to the very large number of ligand combinations compared to the number of proteins that compose the most complex biological liquids. Another reason to explain differences between some of our data and those already reported in the literature (in terms of the types of proteins detected) could be due to the sample processing, that appears to be quite different among the various reports here quoted. For instance, one report14 has evaluated the total loss of number of spots in a 2-D map upon different sample treatments. Taking as a reference urines that had simply been ultrafiltered (and exhibited 829 spots in a 2-D map), various sample precipitation procedure resulted in severe losses. For example, TCA/acetone precipitation gave only 518 spots; acetone precipitation alone resulted in 467 spots and ACN/TFA precipitation gave the lowest number of spots, namely 393. Another report11 gives even more severe losses along the sequence of sample preparation steps. Taken as 100% the protein content of the collected urines, this value diminishes to 94% upon centrifugation and filtration, to 75% upon dialysis, to 27% upon a subsequent lyophilization/dialysis step to end up with only 6% after the last step, precipitation with TCA. Data reported on Figure 5 show that species below about 5000 Da are virtually not present; this is probably due to the precipitation of proteins in a mixture of acetone-methanol (see Material and Method section), prior to trypsin digestion, that does not allow small proteins to precipitate easily. When making an in depth observation of SELDI MS data (the sample is here processed without preliminary precipitation of proteins) it appears that a large number of peptides between 1500 and 5000 can be counted. For untreated urine, the number of species counted here is of 43, while it is respectively 90 and 69 from respective elution with TUC buffer and acidic urea. If redundancies are withdrawn, the net number of species is as high as 115. This number is presumably even higher since in the analysis done in an IMAC-Cu2+ biochip, only the proteic species interacting with this surface can be detected. Although all these species are clearly present they are unavoidably

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ignored when the sample is processed using FT-ICR because of a preliminary protein precipitation operation. According to the various protocols adopted by the various research groups, different classes of proteins could be lost, this resulting in identification of proteins only partly overlapping among the various research groups. Additionally, the fact that the presence of a large number of spots in a 2-D map could be misleading: in most cases, such spots represent string of polypeptide chains, belonging to the same family (isoforms). In other cases, spots scattered through the map could simply be degradation products of a parental protein. A case in point is given in Celis' database:20,21 among the 205 spots listed, 45 were identified as albumin or albumin fragments. Similar results (in terms of albumin fragments) were also reported by Lafitte et al.15 Even when adopting similar techniques the data from single groups offer some variations. Thus, in Pang et al.,9 whereas 1-D-LCMS identifies only 35 proteins, 2-D-LC-MS permits identification of many more (90 species). Yet, five proteins are detected only in 1-D-LC-MS, and not found in a more comprehensive method such as 2-D-LC-MS. Even though 2-D maps permit identification of the lowest number of unique species ( 67 kDa could be filtered through the kidney glomeruli. Yet, all the reports here screened, and adopting 2-D maps for separation, clearly show polypeptide chains up to 100 kDa and above, so that the presence of such large Mr species seems to be a real phenomenon. For a number of those proteins, though, it is conceivable that only fragments are released in urines and thus their presence is inferred by sequencing them in the FT-ICR mass spectrometer. Nevertheless, for many others, it is clear, as demonstrated by 2-D maps, that large species are unambiguously present whose mass may go up to more than 200 kDa.

Conclusions The present data suggest that the combinatorial ligand libraries could be a novel, formidable tool for exploring the hidden, or low-abundance, proteome, i.e., that very large part of the proteome that has escaped detection up to the present time, especially when using mass spectrometry for low and medium molecular masses and 2-D mapping protocols for large proteins. The fact of normalizing the concentration of all species present in solution not only has the advantage of revealing the “deep proteome”, but also has the added benefit of suppressing, during mass spectrometry analysis, the strong signal due to the very abundant proteins. Although we do not know as yet how deep we have been “digging” to bring to the surface the hidden proteome, we feel that, by processing larger amount of biological material and using a greater ligand library diversity, we could explore the proteome of any organism to

further depths, to an extent nobody could have figured out so far. It is believed that, among this large number of unique proteins thus far identified, a few could represent potential novel markers for disease.

Acknowledgment. Supported by MIUR, Roma (PRIN2003, PRIN-2005, FIRB-2001, No. RBNE01KJHT), by Banca Intesa (Milano) and by AIRC (Milan). We thank Gherardo Sabaini for the help in screening literature data and eliminating redundancies from the various data sets reported. L.S. and J.R. gratefully acknowledge Matthias Mann for providing access to DTA supercharge software. References (1) Anderson, L. N.; Anderson, N. G. The human plasma proteome. History, character and diagnostic prospects. Mol. Cell. Proteomics 2002, 1, 845-867. (2) Pieper, R.; Gatlin, C. L.; Makusky, A. J.; Russo, P. S.; Schatz, C. R.; Miller, S. S.; Su, Q.; McGrath, A. M.; Estock, M. A.; Parmar, P. P.; Zhao, M.; Huang, S. T.; Zhou, J.; Wang, F.; Esquer-Blasco, R.; Anderson, N. L.; Taylor, J.; Steiner, S. The human serum proteome: display of nearly 3700 chromatographically separated protein spots on 2D electrophoresis gels and identification of 325 distinct proteins. Proteomics 2003, 3, 1345-1364. (3) Anderson, L. N.; Polanski, M.; Pieper, R.; Gatlin, T.; Tirumalai, R. S.; Conrads, T. P.; Veenstra, T. D.; Adkins, J. N.; Pounds, J. G.; Fagan, R.; Lobley, A. The human plasma proteome. Mol. Cell. Proteomics 2004, 3, 311-326. (4) Edwards, J. J.; Tollaksen, S. L.; Anderson, N. G. Proteins of human urine. III: identification by two-dimensional electrophoretic map positions of some urinary proteins. Clin. Chem. 1982, 28, 941948. (5) Anderson, N. G.; Anderson, N. L. The human protein index. Clin. Chem. 1982, 28, 739-748. (6) Petricoin, E. F.; Zoon, K. C.; Kohn, E. C.; Barrett, J. C.; Liotta, L. A. Clinical proteomics: translating benchside promise into bedside reality. Nat. Rev. Drug Discovery 2002, 1, 683-695. (7) Petricoin, E. F.; Wulfkuhle, J.; Espina, V.; Liotta, L. A. Clinical proteomics: revolutionizing disease detection and patient tailoring therapy. J. Proteome Res. 2004, 3, 209-217. (8) Anderson, L. N.; Anderson, N. G. High-resolution two-dimensional electrophoresis of human plasma proteins. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5421-5425. (9) Pang, J. X.; Ginanni, N.; Dongre, A. R.; Hefta, S. A.; Opitek, G. J. Biomarker discovery in urine proteomics. J. Proteome Res. 2002, 1, 161-169. (10) Pieper, R.; Gatlin, C. L.; McGrath, A. M.; Makusky, A. J.; Mondal, M.; Seonarain, M.; Field, E:; Schatz, C. R.; Estock, M. A.; Ahmed, N.; Anderson, N. G.; Steiner, S. Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on 2D electrophoresis gels with a yield of nearly 1400 distinct protein spots. Proteomics 2004, 4, 1159-1174. (11) Oh, J.; Pyo, J. H.; Jo, E. H.; Hwang, S. I.; Kang, S. C.; Jung, J. H.; Park, E. K.; Kim, S. Y.; Choi, J. Y.; Lim, J. Establishment of a nearstandard 2D human urine proteomic map. Proteomics 2004, 4, 3485-3497. (12) Thongboonkerd, V.; McLeish, K. R.; Arthur, J. M.; Klein, J. B. Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation. Kidney Int. 2002, 62, 1461-1469. (13) Schaub, S.; Wilkins, J.; Weiler, T.; Sangster, K.; Rush, D.; Nickerson, P. Urine protein profiling with surface-enhanced laser-desorption/ionization, time-of-flight mass spectrometry. Kidney Int. 2004, 65, 323-332. (14) Tantipaiboonwong, P.; Sinchaikul, S.; Sriyam, S.; Phutrakul, S.; Chen, S. T. Different techniques for urinary protein analysis of normal lung cancer patients. Proteomics 2005, 5, 1140-1149. (15) Lafitte, D.; Dussol, B.; Andersen, S.; Vazi, A.; Dupuy, P.; Jensen, O. N.; Berland, Y.; Verdier, J. M. Optimized preparation of urine samples for 2D electrophoresis and initial application to patient samples. Clin. Biochem. 2002, 35, 581-589. (16) Smith, G.; Barratt, D.; Rowlinson, R.; Nickson, J.; Tonge, R. Development of high-throughput method for preparing human urine for 2D electrophoresis. Proteomics 2005, 5, in press. (17) Joo, W. A.; Lee, D. Y.; Kim, C. W. Development of an effective sample preparation method for the proteome analysis of body fluids using 2D gel electrophoresis. Biosci. Biotechnol. Biochem. 2003, 67, 1574-1577.


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PR050153R

Exploring the Hidden Human Urinary Proteome via Ligand Library

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