Establishment of a Reliable Method for Direct Proteome

Establishment of a Reliable Method for DirectProteome Characterization of Human ArticularCartilage*Jean-Baptiste Vincourt‡§, Frederic Lionneton‡, Gueorgui Kratassiouk¶,Francois Guillemin¶, Patrick Netter‡, Didier Mainard‡, and Jacques Magdalou‡

Articular cartilage consists mainly of extracellular matrix,mostly made of collagens and proteoglycans. These mac-romolecules have so far impaired the detailed two-dimen-sional electrophoresis-based proteomic analysis of artic-ular cartilage. Here we describe a method for selectiveprotein extraction from cartilage, which excludes proteo-glycans and collagen species, thus allowing direct profil-ing of the protein content of cartilage by two-dimensionalelectrophoresis. Consistent electrophoretic patterns ofmore than 600 protein states were reproducibly obtainedafter silver staining from 500 mg of human articular car-tilage from joints with diverse pathologies. The extractionyield increased when the method was applied to a chon-drosarcoma sample, consistent with selective extractionof cellular components. Nearly 200 of the most intenselystained protein spots were analyzed by MALDI-TOF massspectrometry after trypsin digestion. They represented127 different proteins with diverse functions. Our methodprovides a rapid, efficient, and pertinent alternative topreviously proposed approaches for proteomic charac-terization of cartilage phenotypes. It will be useful fordetecting protein expression patterns that relate patho-physiological processes of cartilaginous tissues such asosteoarthritis and chondrosarcoma. Molecular & Cellu-lar Proteomics 5:1984–1995, 2006.

Articular cartilage is a unique, hypocellular, and avasculartissue, mostly made of extracellular collagens and proteogly-cans (PGs),1 whose physical properties support frictionlessmovements and load-bearing capacity of the articulation (1).Homeostasis of this dense, complex extracellular matrix re-quires perpetual and regulated synthesis/degradation of col-

lagens and PGs by a single cell type, chondrocyte, whichaccounts for only 1% of the volume in human cartilage (2).

Degeneration of cartilage accompanied with partial loss ofarticular function is very common worldwide. The cost of allarthritic diseases together was estimated to be more than 100billion dollars to the United States society for the single year of1997 (3) and is currently increasing due to the aging of humanpopulations in developed countries. In particular, osteoarthri-tis, which is characterized by long term breakdown of thecartilage with no intrinsic inflammatory origin, affects the ma-jority of people over 65 years (4). Yet little medication for itstreatment is available. Analgesic and anti-inflammatory drugsare used for symptomatic treatment of the associated painand inflammation without any effect on the progression of thedisease (4). Therefore it is necessary to better understand,from a molecular point of view, the early events that lead toosteoarthritis and to identify target proteins, which shouldprovide a rationale for the design of new drugs.

Another challenge in the field of cartilage biology is thediagnosis and treatment of malign chondrogenic tumors (5, 6).Chondrogenic neoplasms are a group of tumors that formcartilage because their cells share much of a chondrocytephenotype. “Chondrosarcoma” is a general term used to de-scribe, among chondrogenic neoplasms, those that exhibitmalignancy. Their early diagnosis after histological analysis isvery difficult due to their similarity to benign tumors, which aremuch more frequent (5). Distinction between the two is impor-tant, however, because chondrosarcoma, unlike benign chon-droma, should be treated surgically. It is thus critical to identifyunequivocal molecular markers of chondrosarcoma (5, 6).

In these respects, most efficient strategies aiming at iden-tifying molecular markers and/or pharmacological targets ofcartilage pathologies were based on transcriptional analyses(7–9). These, however, do not directly predict variations ofprotein levels or post-translational modifications. Therefore,direct, 2-DE-based differential proteomics of whole, healthy,and pathological tissues is an attractive alternative (10, 11). Inthe case of cartilage, however, such an approach is difficult toperform (12) due to its high content of PGs and collagens (13).These macromolecules impair isoelectric focusing and maskminor, cellular proteins. Consequently proteomic analysis ofcartilage has so far been performed under either of two re-

From the ‡Laboratoire de Physiopathologie et Pharmacologie Ar-ticulaires, Faculte de Medecine, Unite Mixte de Recherche (UMR)7561 CNRS-Universite Henry Poincare (UHP), 9 Avenue de la Foret deHaye, BP 184, 54505 Vandoeuvre-les-Nancy Cedex, France and¶Centre Alexis Vautrin, Centre de Recherche en Automatisme deNancy UMR 7039, CNRS-Institut National Polytechnique Lorrain-UHP, 54500 Vandoeuvre-les-Nancy, France

Received, February 14, 2006, and in revised form, April 10, 2006Published, MCP Papers in Press, May 9, 2006, DOI 10.1074/

mcp.T600007-MCP2001 The abbreviations used are: PG, proteoglycan; CPC, cetylpyridin-

ium chloride; 2D, two-dimensional; 2-DE, two-dimensional electro-phoresis; CV, coefficient of variation.

Technology

© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.1984 Molecular & Cellular Proteomics 5.10This paper is available on line at http://www.mcponline.org

by guest on January 1, 2019http://w

ww

.mcponline.org/

Dow

nloaded from

http://www.mcponline.org/

strictive conditions. The one consists of cartilage enzymaticdigestion followed by monolayer primary culture of chondro-cytes in fetal calf serum (14), which are likely to introduceartifactual gain or loss of differentiation markers (15–17) anddo not clarify changes in the extracellular matrix. The other isbased on analysis of proteins secreted by cartilage explantsinto culture medium (13). It is technically complex, requiresmuch starting biological material, and allows detection of onlya minority of cartilage constituents.

To provide a more representative map of the cartilage pro-teome, we developed a method allowing selective extractionof proteins directly from human articular cartilage and theirsubsequent resolution by 2-DE. This novel strategy allowedus to generate sufficient amounts of protein extracts from 500mg or less of macroscopically normal cartilage and to resolveat least 600 spots on 2D gels after silver staining. Extractionwas performed efficiently and reproducibly from varioussources of pathological tissues from several joints. Interest-ingly proteins were extracted more efficiently when themethod was applied to a chondrosarcoma sample presum-ably due to the higher cellularity of this tissue. MS analysis of191 spots revealed a broad diversity in the protein identity andfunction. This protocol provides a powerful alternative to pre-vious strategies aiming at characterizing cartilage phenotypesat a molecular level. It will be especially helpful at achievingdifferential analysis of cartilage in physiopathological condi-tions for identification of protein states that are altered as acause or a consequence of the diseases.

EXPERIMENTAL PROCEDURES

Tissue Procurement and Processing—This study was performed inconformity with the declaration of Helsinki principles. Articular carti-lage was obtained from Service d’Orthopedie Chirurgicale (CentreHospitalier Universitaire, Nancy, France) in the context of amputationand prosthesis replacement. The chondrosarcoma sample was ob-tained from the same source in the context of tumor amputation. Thetumor was diagnosed and graded by the Service d’Anatomie etCytologie Pathologique from Centre Hospitalier Universitaire, basedon radiological and histological analysis. Written informed consentwas given by the patient. Cartilage samples were kept in cold salinefor transportation and immediately processed. Macroscopically nor-mal cartilage of various pathological sources (Table I) was dissectedand washed extensively in cold Dulbecco’s PBS with eventual curet-tage for removal of all blood cells, wiped on paper towels, and frozenat �80 °C until further use. For chondrosarcoma, regions with mac-roscopically visible vasculature were removed to avoid contaminationby endothelial and blood cells.

Protein Extraction and Preparation—Cartilage was soaked withcartilage extraction buffer (500 mM NaCl, 50 mM HEPES, pH 7.2,Complete protease inhibitor (Roche Applied Science)) and frozen ona cryotome plate (HM440E, Microm) at �30 °C. The tissue was cutinto 10-�m slices and suspended in cold extraction buffer at 100 mgof cartilage/ml. Samples were agitated at room temperature for 1 h.Extracted material was clarified upon centrifugation (6,000 � g for 5min), and 1% (w/v) CPC was added. PGs and CPC were allowed toaggregate (1 h at room temperature under agitation) and removed bycentrifugation (6,000 � g for 5 min). The resulting supernatant wassubmitted to chloroform/methanol precipitation (18) with a slightmodification. After the first centrifugation (14,000 � g for 30 min),

both liquid phases were removed. The insoluble material was resus-pended in 4 volumes of methanol and centrifuged again in identicalconditions. Pellets were resuspended in 1 volume of methanol, trans-ferred to safe lock Eppendorf tubes, spun down, dried, and resus-pended in 200 �l of 2D Sample Solution (2DSS; 7 M urea, 2 M thiourea,4% (w/v) CHAPS, 0.2% (w/v) Triton X-100, Complete). For proteinconcentration measurement, samples diluted in a final volume of 10�l of 2DSS were acidified with 2 �l of 0.1 N HCl and processed forBradford assay (19) using bovine serum albumin solubilized in 2DSSas a standard. Protein samples were diluted to 1 �g/�l in 2DSSsupplemented with 1% (w/v) DTT and trace bromphenol blue, ali-quoted at 200 �g/sample, and kept frozen at �80 °C until further use.

Two-dimensional Electrophoresis—Cartilage protein samples (200�g) were thawed and agitated for 2 h, ultracentrifuged (100,000 � gfor 30 min at room temperature) for insoluble material removal. Thesupernatant was allowed to rehydrate IPG strips (Bio-Rad, 24 cm, pH3–10, 10 h at room temperature) in a final buffer volume of 450 �l (2DSSsupplemented with DTT and bromphenol blue). Electrofocalization wasperformed in a Protean IEF cell (Bio-Rad) through a single step programaccording to the manufacturer’s recommendations. Strips were cut oneach side for obtaining strips of pH range 4.5–8. Disulfide bonds ofproteins were reduced by 10-min incubation in 4 ml of equilibrationbuffer (6 M urea, 2% SDS, 50 mM Tris, pH 8.8, 20% (v/v) glycerol)containing 130 mM DTT. Free sulfhydryl groups were alkylated by treat-ment with 135 mM iodoacetamide for 10 min in equilibration buffer.Strips were transferred to the top of 12% (w/v) acrylamide denaturinggels poured in Criterion cassettes (Bio-Rad) and run at 17 mA.

Gel Staining—Gels were fixed for 2 h in two successive baths of30% (v/v) methanol, 10% (v/v) acetic acid. For silver staining, gels wereextensively washed in water overnight. Gels were sensitized for 2 min in0.02% (w/v) sodium thiosulfate, quickly washed twice in water, andimpregnated in 0.2% (w/v) silver nitrate for 1 h. Excess silver nitrate waswashed quickly, and staining was developed with 3% (w/v) potassiumcarbonate, 2.5 mg/liter sodium thiosulfate, 0.025% (v/v) formalin andstopped with 1% (w/v) acetic acid for 30 min. Gels were washed inwater and kept in water at 4 °C. For colloidal Coomassie Brilliant Bluestaining, gels were washed several times in fixative and incubated instaining solution (8% (w/v) ammonium sulfate, 1.6% (w/v) phosphoricacid, 0.08% (w/v) Coomassie G250) overnight. The next day, gels werewashed extensively in water until optimal contrast was obtained.

Reproducibility Assay—Extraction of proteins was performed threetimes independently in three successive experiments, each from 500mg of the same cartilage sample. Protein concentration of eachextract was quantified, and 200 �g were used for 2-DE. Gels werestained with silver nitrate and scanned. Qualitative and quantitativecomparison of triplicates was performed using the PDQuest software(Bio-Rad) on a relatively central zone of the gels to avoid board effectsand poor resolution in the pI dimension. For qualitative assay, thesoftware was used in the automatic mode with subsequent manualcorrection. For semiquantitative comparison, integrated optical den-sities of automatically detected, qualitatively conserved spots werenormalized to the total density of validated spots from the corre-sponding gel. The normalized coefficient of variation (CV) of each spotwas calculated, and the percentage of spots that fall within a definedCV cutoff value (ranging from 0.3 to 1) is reported in Table II.

Trypsic Digestion—After excision, spots (2-mm diameter) wereprocessed. All washes were performed in 30 �l for 15 min. Spots werewashed twice in water and twice in fresh 50 mM ammonium bicar-bonate, 50% (v/v) acetonitrile. Spots were dehydrated for 1 min in100% (v/v) acetonitrile, washed once again in 50 mM ammoniumbicarbonate, 50% (v/v) acetonitrile, and dried in a SpeedVac(RC10.10, Jouan) for 15 min. Each sample was digested overnight at37 °C in 7 �l of sequencing grade modified trypsin (Promega) solution(12.5 ng of trypsin/�l in 25 mM ammonium bicarbonate).

Direct Proteomic Analysis of Human Cartilage

Molecular & Cellular Proteomics 5.10 1985


ww

.mcponline.org/

Dow

nloaded from


Mass Spectrometry—After digestion, the trypsin solution contain-ing peptides was collected. For further peptide extraction, 8 �l of 50%(v/v) ACN, 2% (v/v) TFA were added onto each spot and sonicated for10 min in a waterbath sonicator (Transsonic 570, Elma). ACN (100%,v/v) was added (2 �l). Both solutions containing peptides were pooledtogether. Samples (0.6 �l) were laid on a MALDI target (AnchorChip,Bruker Daltonik) using the dried droplet method with �-cyano-4-hydroxycinnamic acid as a matrix. Peptides were analyzed by MALDI-TOF mass spectrometry using an Ultraflex mass spectrometer(Bruker Daltonik). Measurements were externally calibrated with dif-ferent peptide standards. When required, spectra were internally cal-ibrated with peptides resulting from trypsin autolysis. Monoisotopicpeptide masses were assigned, and searches were performed usingthe MASCOT software (Matrix Science, Oxford, UK) with carboxam-idomethylation of cysteines as fixed modifications and methionineoxidations as variable modifications searching the Mass Spectrome-try Protein Sequence Database (MSDB) and Swiss-Prot (www.ex-pasy.ch/sprot/) databases with a mass tolerance of 50 ppm.

RESULTS

Establishment of a Method for Proteome Extraction andAnalysis from Human Arthritic Articular Cartilage—Intrinsiclimitations of classical proteomic approaches for studyingcartilage are mostly due to the high content in this tissue ofPGs and collagen species, which strongly affect isoelectricfocusing (13) and impair detection of minor proteins. PGs andcollagens, however, have particular biochemical properties,based on which we could design a method allowing theirexclusion in an exhaustive protein extraction system.

First because cartilage is solid, protein extraction requiresdisassembling of the tissue to allow its impregnation by thechosen buffer. This is classically done by total dissociation ofthe tissue (20). On the contrary, we preferred to preserve theinitial structure of the matrix as much as possible to minimizemechanical extraction of collagens and PGs. For this purpose,cartilage was dissected through cryotome section into 10-�mslices.

Collagen molecules assemble into large, macroscopic, in-soluble fibers depending on multiple, well organized hydrogenbonds (21). As such, their cohesion is strongly affected bychaotropic agents, such as urea, which is classically used forprotein solubilization prior to 2-DE analysis (Fig. 1A, rightlane). In contrast, buffers containing high NaCl concentrationare relatively permissive for collagen fibers cohesion whileallowing “solubilization” of most other protein components ofcartilage (Fig. 1A, left lane). The obtained extract was notresolvable by 2-DE presumably due to its high remaining PGcontent (Fig. 1B). We took advantage of the previously de-scribed method for removal of PGs by selective precipitationwith CPC (13), which we found to be efficient at NaCl con-centrations not exceeding 500 mM. Finally because CPC is anionic detergent, it should be removed from the extract as wellas salts as their presence would impair isoelectric focusing.For this purpose, we purified the resulting protein extract bymethanol/chloroform precipitation (18) at room temperature,which also removes nucleic acids and lipids. The resultingpellet was dried and solubilized in a classical 2-DE loading

buffer, providing a protein extract that was efficiently resolvedby 2-DE, as shown on Fig. 1C. A standardized method forcartilage proteomic analysis, described in detail under “Ex-perimental Procedures,” was designed based on these findings.

Efficient Proteome Extraction from Cartilage of Various Or-igins and Pathological States—Because such a method hasmany potential uses in studying cartilage biology, we assayedits capability on various kinds of human cartilaginous tissues.As shown in Table I, these include articular cartilage from hip,knee, and talus affected by various diseases. Also a sample oflow grade chondrosarcoma was included. Cartilaginous tu-mors of low grade possess the same intrinsic incompatibilitieswith classical proteomic approaches as articular cartilage be-cause they synthesize a comparable extracellular matrix (5,22). Consequently biochemical methods that apply to articularcartilage should also apply to such tumors. All samples wereprocessed identically and provided extracts that could be wellseparated by 2-DE (Fig. 2). Amounts of protein extract ob-tained from all articular cartilage samples were very similar(see Table I) with an average extraction yield higher than 400�g of extract/g of cartilage. Also the extraction efficiency wasfound to be reproducible (S.D. � 10%) when performed twiceindependently on the same cartilage sample (data not shown).When applied to a chondrosarcoma sample, the method wasdramatically more efficient as its extraction yield reached 2.4 mgof proteins/g of tissue. Because the major macroscopically vis-ible difference between cartilaginous tumor and articular carti-lage is a higher cellularity of chondrosarcoma (22), it is likely that(i) the extraction yield increases with cellularity and (ii) intracel-lular proteins occupy a high proportion of the extract.

Reproducible Extraction of Proteins from Cartilage Sam-ples—Extracts obtained with this method exhibited a rela-tively similar electrophoretic pattern, in the sense that mostspots appeared identical from one sample to another, accord-ing to their respective shapes and coordinates on 2D gels (Fig.2). Reproducibility is a recurrent issue when using 2-DE be-cause 2-DE itself introduces substantial variation in the num-ber, intensity, and localization of spots on 2D gels (23, 24). Tochallenge the reproducibility of the proposed method, two ofthe samples shown in Fig. 2 were subjected to independent,triplicate extractions, 2-DE, silver staining, and subsequentcomparative analysis. We used an osteoarthritic cartilagesample and the chondrosarcoma sample (Fig. 2, B and F). Foreach tissue, reproducibility of the results was assessed qual-itatively and quantitatively (Table II). The spot counts were558 � 39 for sample S9 and 613 � 22 for sample S6, showinglittle variation in this respect. Spots that could not be matchedfrom one gel to another (from the same sample) were countedand found to represent 18%, on average (Table II), of all spotscounted from the corresponding gels. This value was close tothose reported for the reproducibility of 2-DE using silverstaining detection (25) and other comparable methods (23),showing good qualitative repeatability of the method. To eval-uate the quantitative reproducibility, the content of each con-


1986 Molecular & Cellular Proteomics 5.10


ww

.mcponline.org/

Dow

nloaded from


served spot was estimated as described under “ExperimentalProcedures.” For each spot, the CV of the quantity, as meas-ured between all three gels, was calculated. Spots were clas-

sified as populations that have a CV falling within a definedcutoff value (ranging from 0.3 to 1, Table II) as suggestedpreviously (23). According to Reed et al. (26), the repartition of

FIG. 1. Principles of cartilage proteome extraction method. A, comparison of extraction buffers. Human knee arthritic cartilage (100 mg)was washed in saline and subjected to cryotome section and resuspension for 1 h in 500 �l of extraction buffer containing 50 mM HEPES, pH7.2, and either 500 mM NaCl or 2 M urea, 4% (w/v) CHAPS. After removal of insoluble material by centrifugation at 6,000 � g for 5 min, 125�l of 5� Laemmli sample buffer was added. Samples were boiled, and 50 �l were separated on a 10% (w/v) acrylamide denaturing gelsubsequently stained with Coomassie Brilliant Blue. Arrows indicate expected molecular masses of mature collagen II and aggrecan. B andC, cartilage proteins were extracted in 500 mM NaCl-containing buffer as described in A. In B the protein content was directly precipitated withmethanol/chloroform, whereas in C PGs were first aggregated with 1% (w/v) CPC according to the “Experimental Procedures,” and the proteincontent was subsequently precipitated with methanol/chloroform. Extracts were then solubilized in 2DSS and assayed for protein concen-tration, and 200 �g were processed for 2-DE. Silver staining was performed.




ww

.mcponline.org/

Dow

nloaded from


CV values found in our study allows searching for 4-foldexpression differences between two samples with as little as1.6% false positive spots. Therefore, using populations ofsamples, the proposed procedure is suitable for the identifi-cation of protein markers of cartilage diseases.

Toward Exhaustive Proteomic Analysis of Articular Carti-lage—One important criterion for defining the ability of themethod to enlighten cartilage phenotypes is the variety inidentity and function of analyzed proteins. As an initial effort toemphasize heterogeneity of the extract, 220 spots were ran-

TABLE ICartilage samples used in this study

For each sample, gender (F, female; M, male), age, and pathology of the patient are given. Also extraction yield of the method, as estimatedby the acidified Bradford assay, is annotated.

Sample reference Gender Age at sampling Original tissue Disease Protein extraction from 1 g

yr �g

S9 F 69 Hip Arthritis 480S2 M 61 Hip Psoriasic arthritis 430S3 F 73 Hip Arthritis 390S4 M 54 Knee Osteonecrosis, early osteoarthritis 380S6 M 29 Upper tibia Chondrosarcoma, grade I 2,400S8 M 53 Talus Rheumatoid arthritis 380

FIG. 2. Electrophoretic profiles of cartilage proteomes from various tissues. Protein extracts (200 �g) from various tissues (see Table I)were loaded on 2D gels. 2-DE analysis was performed as indicated under “Experimental Procedures.” A, S3; B, S9; C, S2; D, S4; E, S8; F, S6.




ww

.mcponline.org/

Dow

nloaded from


domly chosen among those of highest staining for mass spec-trometry analysis after tryptic digestion. For ease of proce-dure, this was done from a sample of femoral arthriticcartilage after colloidal Coomassie Brilliant Blue staining (Fig.3). The content of several spots could not be identified despite

detection of normal mass spectra by MS presumably due toabundant glycosylations. The content of 191 spots, however,was unambiguously identified and annotated on Fig. 3. Alsoidentified proteins were classified based on their known phys-iological functions in Table III. Those identified proteins cor-

FIG. 3. 2D map of the human articular cartilage proteome. Protein extract (450 �g) from sample S3 was separated by 2-DE as describedunder “Experimental Procedures” and stained with colloidal Coomassie Brilliant Blue. The content of 191 spots was identified by MS aftertryptic digestion. Spots are indicated by arrows, and their names are abbreviated. Detailed analysis is compiled in Table III.

TABLE IIQualitative and quantitative assessment of the reproducibility

Extraction was performed in separate triplicates from samples S9 and S6 (see Table I). Resulting gels were scanned and analyzed usingPDQuest software as mentioned under “Experimental Procedures.” Analysis was restricted to a relatively central area of the gels to avoid boardeffects and poor resolution in the pI dimension. Spots were detected automatically and corrected manually. They were counted and matchedbetween gels. The number of detected spots is indicated as mean � S.D. Unmatched spots are expressed as their average percentage relativeto total spot numbers � S.D. For quantification, spots common to all triplicates within each group were quantified and normalized to the totaldensity of validated spots from the corresponding gel. For each spot, the CV between all three gels was calculated. The proportion of spotswith a CV lower than the indicated cutoff is shown in the corresponding column as a percentage.

Tissuesample

Qualitative Quantitative (spot percentage with CV lower than the cutoff)

Spot count ininvestigated area

Gel-to-gelunmatched spots 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

%

S9 558 � 39 18 � 2 41 59 72 83 91 96 98 99

S6 613 � 22 18 � 5 59 75 84 92 97 98 99 99




ww

.mcponline.org/

Dow

nloaded from


TABLE IIIFunctional classification of proteins identified by MS

Classification was performed according to the Swiss-Prot database. Abbreviations used for annotation of the proteomic map in Fig. 3 arelisted. The General Identifier (GI) from the National Center for Biotechnology Information (NCBI) and full name of the corresponding protein areindicated as well as its theoretical molecular weight/pI. Also the number of peptide masses isolated from the corresponding spot that matchthe reference sequence and their coverage of the sequence are annotated. ER, endoplasmic reticulum; EGF, epidermal growth factor; HBsAg,hepatitis B surface antigen; TGF�, transforming growth factor �; HS, heparan sulfate.

Abbreviation NCBI GIa Full name of closest identified proteinTheor. molecular

weight/pIaPept. no./

seq. cov. (%)b

Metabolism and energyADK 13097732 Adenosine kinase, isoform a 39,112/6.23c 16/52ADH 24497577 Aldo-keto reductase family 1, member A1 36,761/6.34c 15/54AR7A2 39645003 Aldo-keto reductase family 7 member A2 39,889/6.74 12/37ALDR 113596 Aldose reductase, aldehyde reductase 36,028/6.51c 10/47ENOA 1167843 �-Enolase 47,350/6.99 14/49

11/3520/56

CADH 515147 Carbonic anhydrase I 28,408/6.90 12/59ALAD 248839 �-Aminolevulinate dehydratase 36,758/6.16 14/38LDHb 126041 L-Lactate dehydrogenase B chain 36,769/5.72 14/40NNMT 730163 Nicotinamide N-methyltransferase 30,011/5.56c 11/47PGAM-1 130348 Phosphoglycerate mutase isozyme B 28,769/6.75 15/70

14/66PNP 55925942 Purine-nucleoside phosphorylase 32,325/6.45 15/58PYK 20178296 Pyruvate kinase, isozymes M1/M2 58,339/7.95 19/47TPIS 39932641 Triose-phosphate isomerase 26,807/6.51 10/61

13/7513/75

Protein synthesisACP1 2134737 �-Complex protein 1 37,987/6.66 16/57EF1B 119163 Elongation factor 1-� 24,788/4.50 9/41LAMB 34234 Laminin-binding protein 31,888/4.84c 13/50BAT1 2500529 Nuclear RNA helicase (DEAD family) BAT1 49,416/5.44 16/46EF1g 39644794 Translation elongation factor 1� 50,156/6.27 13/37

Protein fate (maturation,degradation, and trafficking)

Grp94 119360 94-kDa glucose-regulated protein 92,696/4.76 21/25AHC 20141702 Adenosylhomocysteinase 48,255/5.92 12/28CALR 1905911 Calreticulin 48,283/4.29 10/31CALU 14718453 Calumenin 37,198/4.47 11/40GroEL 107086 Chaperonin GroEL precursor 61,187/5.70 14/41HSCP70 87625 dnaK-type molecular chaperone, HSCP70 71,082/5.37 22/41ELI 266344 Elastase inhibitor 42,829/5.90c 11/41ER46 29839560 Endoplasmic reticulum protein of 46 kDa 48,283/5.63 11/32ER28 7512406 Endoplasmic reticulum luminal protein 28 29,032/6.77 11/51Grp78 6470150 ER Ca2�-binding protein grp78 71,002/5.23 26/44HSP90 20149594 Heat shock 90-kDa protein 1, � 83,423/4.97 18/31APG2 4579909 HSP110 family, member apg-2 95,096/5.18 14/24PA2G4 33879698 PAG2G4 metallopeptidase 41,996/7.14 17/53CATB 181192 Preprocathepsin B, fragment 38,752/5.88c 10/30P4HA2 20455169 Prolyl 4-hydroxylase �2 subunit precursor 60,994/5.50 15/36PSD13 20978558 Proteasome 26 S subunit 13, non-ATPase 43,175/5.44 12/38PSA5 P28066 Proteasome subunit � type 5 26,565/4.74 11/56PDI 860986 Protein-disulfide isomerase 57,480/4.76 11/31

19/4318/4418/42

PI9 1709896 Proteinase inhibitor 9 43,004/5.61c 11/37RCN3 30316268 Reticulocalbin-3 precursor, RLP49 37,470/4.74 9/35RBPS 2460318 RNA-binding protein regulatory subunit 20,050/6.33 11/65S13L 50403748 SEC13-related protein, SEC13-like protein 1 35,900/5.22 7/35STIP1 400042 Stress-induced-phosphoprotein 1 63,227/6.40 13/24TCP1t 9988062 T-complex protein 1, � subunit 60,022/5.42 15/34




ww

.mcponline.org/

Dow

nloaded from


TABLE III—continued



seq. cov. (%)b

TERA 2984586 Transitional ER ATPase 89,819/5.14 25/32OTUB1 44888286 Ubiquitin thioesterase protein 31,493/4.85 8/43

Cytoskeleton and cell structuregACT 12653055 Actin, �1 42,108/5.31 21/68ACTN1 46397817 �-Actinin cytoskeletal isoform 103,480/5.22c 25/35CAPa 7448799 Capping protein � subunit isoform 1 33,073/5.45 12/61

9/49EZR 21614499 Ezrin 69,313/5.94 18/32FASN 2498357 Fascin, 55-kDa actin-bundling protein 54,992/6.81 16/37GELS 4504165 Gelsolin isoform a 85,925/5.77 13/25LASP1 2135552 Lasp-1 protein 30,185/6.11 11/32

11/36TPM 88926 Tropomyosin 26,618/4.77 10/37TPM3 15929959 Tropomyosin 3 27,386/4.77 12/41TPM4a 54039751 Tropomyosin �4 chain 28,487/4.67 11/42

8/32TUBA1 17921989 Tubulin, �1 50,626/4.97 11/36TUBB 29788785 Tubulin, � polypeptide 48,135/4.70c 9/28VIM 57471646 Vimentin 49,680/5.19 13/24

13/3413/2712/2727/559/27

28/5528/5628/5625/559/25

17/3226/5320/40

VCN 340237 Vinculin 117,220/5.83 28/28Cell defense

eSODM 134635 Extracellular superoxide dismutaseprecursor

26,321/6.14c 7/36

GST 284131 Glutathione transferase 23,569/5.43c 11/55GSTO1 6016173 Glutathione transferase Omega 1 27,704/6.77c 10/34HSP27 662841 Heat shock protein 27 22,427/7.83c 8/42PRDX6 1718024 Peroxiredoxin 6 25,002/6.02c 13/74PGTP 404108 Plasma glutathione peroxidase 16,760/8.93c 9/54SODM 134665 Superoxide dismutase mitochondrial 22,705/7.53 7/42

Signal transduction1433e 3493336 14-3-3 � 29,326/4.63 14/501433g 48428721 14-3-3 protein � 28,325/4.80 13/501433t 112690 14-3-3 protein � 28,032/4.68 14/551433z 52000887 14-3-3 protein /� 27,899/4.73 13/6440PK 23879 40-kDa protein kinase 40,794/6.67 10/32ANX1 4502101 Annexin I 38,918/6.57 13/52

8/3517/60

ANX2 50845386 Annexin II 38,808/7.57 17/5719/629/31

10/31ANX4 4502105 Annexin IV 36,290/5.84 24/68

17/51ANX5 468888 Annexin V 35,971/4.94 21/66

8/2919/61




ww

.mcponline.org/

Dow

nloaded from





seq. cov. (%)b

CP9s4 38373690 COP9 signalosome subunit 4 46,525/5.57 10/30DRP2 3122051 Dihydropyrimidinase-related protein 2 62,711/5.95c 25/62GDI2 56410846 GDP dissociation inhibitor 2 51,087/6.11 13/43KRap 12643951 Serine-threonine kinase receptor-associated

protein38,756/4.98 10/46

SPD1 52783472 Spondin-1 precursor 93,537/5.85 13/23TCTP 136479 Translationally controlled tumor protein 19,697/4.84 9/44

Unknown intracellular functionsTRP14 33415057 Transformation-related protein 14 43,248/5.49 17/41WDRP 62897353 WD repeat-containing protein 1 isoform 1

variant66,822/6.17 15/35

Extracellular matrixACTR 177933 �1-Antichymotrypsin precursor 45,567/5.32 14/41a2PG 4502005 �2-HS-glycoprotein 40,098/5.43 8/23CACP1 47777317 Cartilage acidic protein 1 70,404/5.09 11/23COL2 825645 Pro-� (II) collagen 31,919/6.35 11/44COMP 27530066 Cartilage oligomeric matrix protein 85,403/4.34 24/41

11/26LUM 20141464 Keratan sulfate proteoglycan lumican

precursor38,747/6.16c 9/34

9/31KNG 28932976 Kininogen 48,936/6.29c 13/34

13/3713/29

MAT3 2326547 Matrilin-3 34,944/5.55c 9/5012/70

MMP3 4505217 Matrix metalloproteinase 3 preproprotein 54,229/5.61 13/24OIF 107244 Osteoinductive factor 34,243/5.46 9/40

9/409/409/458/42

PRELP 1709586 Proline-arginine-rich end leucine-rich repeatprotein

44,181/9.47c 9/27

14/37PGLP 14585875 Proteoglycan link protein 40,767/7.10 10/38HTRA1 18202620 Serine protease HTRA1 precursor 52,167/8.09 11/27BIGH3 2498193 TGF�-induced protein BIGH3 precursor 75,261/7.62c 24/39TGFi68 62897225 TGF�-induced, 68-kDa variant 75,231/7.62 14/22PCPE 6919941 Type 1 procollagen C-proteinase enhancer

protein48,797/7.41 13/34

15/44Complement components

C1r 115204 Complement C1r subcomponent precursor 81,661/5.89 20/42C1s 115205 Complement C1s subcomponent precursor 78,174/4.86 17/26

21/37C3p 116594 Complement C3 precursor 188,585/6.02 21/15

16/1339/24

C6p 105741 Complement C6 precursor 108,367/6.39 16/21C8p 29575 Complement C8-� propeptide 63,605/8.24 18/39CFB 584908 Complement factor B precursor 86,847/6.67 20/36CFH 62739186 Complement factor H isoform a precursor 143,710/6.28 44/40

Synovial fluid components(classically known asplasma components)

aFP 178236 �-Fetoprotein 68,354/5.67 23/47IgKF 3721651 Anti-HBsAg immunoglobulin Fab chain 23,783/8.30c 7/55ATHR 28907 Antithrombin 47,415/6.25c 14/35




ww

.mcponline.org/

Dow

nloaded from


respond to 127 distinct gene products. The ratio between thetwo (127:191) denotes respectable variety of proteins in theextracts. Eighty-two of these have intracellular roles, rangingfrom general cellular physiology to signal transduction, stressresponse, and matrix processing. We also found 16 proteinsfrom the extracellular matrix, mostly in an unprocessed, intra-cellular form. Seven complement factors were identified, in-cluding both premature and processed forms, in agreementwith previous demonstration of their expression by chondro-cytes in vivo (27). In total, 105 identified proteins actuallyoriginated from inside the chondrocytes, confirming a selec-tive extraction of intracellular proteins. Finally 22 proteins arecommonly considered as plasma components, yet synovial

fluids were previously shown to contain those proteins as well(28). Because synovial fluids do communicate directly withcartilage, unlike plasma, we classified them as “synovial fluid”components.

DISCUSSION

Despite an increasing need for large scale characterizationof the cartilage proteome (29), classical and non-classicalproteomic approaches for cartilage have been impracticaluntil recently (12) due to the particular content of this highlyspecialized tissue (13). Indirect approaches have been usedconsisting of the analysis of proteins secreted by cartilagewhen incubated in culture medium (13) or proteins extracted




seq. cov. (%)b

ATHR3 179130 Antithrombin III 53,025/6.32c 19/409/24

APOA1 490098 Apolipoprotein AI 28,061/5.27 15/6114/5511/51

APOA4 178779 Apolipoprotein A-IV precursor 43,358/5.22 21/58APOH 6573461 �2-Glycoprotein-I, apolipoprotein H 37,499/8.37c 9/45

9/45EFEM1 15072400 EGF-containing fibulin-like

extracellular matrix protein 156,885/4.95 21/51

EDIL3 21410823 EGF-like and discoidin I-like domainsprotein 3

55,128/7.08 11/30

11/3013/34

FBGb 399492 Fibrinogen � chain precursor 49,768/7.02c 29/61FBGd 2781208 Fibrinogen fragment d 36,331/7.66 29/81HPX 1335098 Hemopexin 49,948/6.43c 16/53

14/39HP 47124562 Haptoglobin protein 31,647/8.48c 15/45

14/4214/4314/43

ZAG 7246026 Human zinc-�2-glycoprotein 30,775/6.03c 14/55IgKL 21669345 Immunoglobulin light chain 28,918/6.15c 8/51PEDF 15217079 Pigment epithelium-derived factor 46,484/5.97 17/44

19/5118/50

CPM 180256 Preceruloplasmin 122,983/5.44 18/23ATRY 15080499 Serine proteinase inhibitor, antitrypsin 46,864/5.36 18/51ALB 3212625 Serum albumin 67,931/5.69 12/22

22/4515/3512/2818/3615/30

TTN 37409 Tetranectin 20,480/5.80c 5/37TFR 339453 Transferrin precursor 79,280/6.81 32/51

28/44VDBP 32483410 Vitamin D-binding protein precursor 54,526/5.40 17/55a NCBI GI and molecular weight/pI are given for the closest isoform of the identified protein.b Peptide number and sequence coverage were calculated based on the reference sequence.c Indicates that none of the spots from which the reference protein was found has the corresponding molecular weight/pI coordinates on the

map.




ww

.mcponline.org/

Dow

nloaded from


from chondrocytes in culture (14).Here we describe a novel method allowing direct 2D gel-

based proteomic characterization of cartilaginous tissues.This method was found to be qualitatively and quantitativelyreproducible (Tables I and II) using articular cartilage of sev-eral joints affected by some of the most common cartilage-related pathologies, and extracted content could be well sep-arated by 2-DE (Fig. 2). The method was also found to bepertinent to the analysis of cartilaginous tumors. Electro-phoretic profiles of more than 600 protein spots were allglobally similar, providing a “cartilaginous tissue signature” inthe sense that major spots were identically present on stainedgels made from any sample.

A reference sample was analyzed by MS, revealing theidentity of 191 of its most intensely stained spots seen on gel.The extracted proteins had very broad spectrum of physio-logical functions, most being intracellular. Indeed the electro-phoretic pattern of extracts described here shared much ho-mology with that of the previously described proteome ofcultured chondrocytes (14). This similarity appears strikinglywhen comparing lists of identified proteins: 41 of 93 proteinsidentified by Ruiz-Romero et al. (14) were also found in ourstudy (Table III). Our approach, however, presents severalmajor advantages. From a practical point of view, our methodallows the use of frozen, eventually non-sterile cartilage sam-ples. This is obviously impossible for alternatives using cellculture; also it is faster and requires no specific material. Moreimportantly, our method provides direct analysis of the in vivostatus of chondrocytes. Cultivation of chondrocytes as mono-layer in fetal calf serum-containing media is a very importanttool for studying molecular mechanisms of their physiology.When possible, however, its use should be avoided in thecontext of detailed expression studies as it introduces bothvariability and loss of differentiation markers (15–17). In thissense, our protocol constitutes much of a conceptual im-provement compared with those that involve chondrocyteculture. One possibly direct consequence of this improvementis the finding of several complement factors originating fromchondrocytes (27). These are not only obvious regulators ofthe immune system but also apparent modulators of cartilagematrix degradation. As such, they have potential involvementin cartilage degenerative diseases and are interesting to betaken under consideration (summarized and discussed inRefs. 27, 30, and 31)).

There is one single precedent of a method allowing directproteomic analysis of cartilage (13) based on “secretion” of denovo synthesized proteins from cartilage explants into culturemedium. This work first described the principle of PG removalby CPC precipitation. However, because this study was fo-cused on diffusing proteins, cellular components were poorlyrepresented; only 27 different proteins were analyzed, most ofwhich corresponded to cartilage matrix components and reg-ulatory molecules. Indeed the authors argued that proteinsoriginating from synovial fluids should not be taken into ac-

count because they are not produced by chondrocytes. Onthe contrary, we considered them as taking part of the generaland pathological physiology of cartilage and, as such, poten-tially informative. Additionally intracellular proteins are muchbetter represented with our method because it was designedfor their targeted extraction. As a result, our approach pro-vides a broad and detailed look at molecular events that takeplace in cartilage.

The most obvious limitation of our protocol is the largeamount of albumin remaining in purified extracts (Fig. 3, es-timated to 30%). This results in masking of other proteins ofsimilar molecular weight/pI and reduces the quantity of in-formative proteins to be loaded on isoelectric focusing strips.For some applications, users might want to include an addi-tional step of immunoaffinity-based removal of albumin as isclassically done for plasma samples (32). However, we foundit difficult to remove more than 80% of the albumin content.Also removal of albumin seemed to be a cause of quantitativevariability for certain protein spots (unidentified, data notshown). Furthermore albumin was not found at such a highlevel from all kinds of cartilaginous tissues (see Fig. 2). Inparticular, albumin depletion might be avoided in the contextof cartilaginous tumor characterization, which we are cur-rently conducting without major difficulties in this regard.

In summary, our newly set-up protocol combines advan-tages of both previously proposed methods for global carti-lage proteome analysis together with additional ones. It isachievable from low quantities of starting material, a majorimprovement, particularly for studying cartilage tissue fromsmall joints and tumors (Fig. 2, E and F). Also it allows con-venient practices, such as sample freezing prior to process-ing. This procedure was found to be reproducible from severalsources of articular cartilage differing in joint origin and path-ological states. This method will be useful for the develop-ment of proteomic strategies aiming at defining key molecularevents of cartilage pathologies.

Acknowledgments—We thank Dr. M. Ouzzine and Dr. L. Grossin,from our laboratory, and S. Shaw, from NCI, National Institutes ofHealth, Bethesda, MD, for careful reading of the manuscript andconstructive comments.

* This work was supported in part by the Association pour laRecherche contre le Cancer (ARC), Ligue Regionale contre le Cancer,Region Lorraine, and Institut Federatif de Recherche 111 Bioingenieu-rie. The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

§ Supported by ARC. To whom correspondence should be ad-dressed. Tel.: 33383683950; Fax: 33383683959; E-mail: [email protected].

REFERENCES

1. Muir, H. (1995) Biomechanics, structure, function and molecular biology ofcartilage matrix macromolecules. Bioessays 17, 1039–1048

2. Huber, M., Trattnig, S., and Lintner, F. (2000) Anatomy, biochemistry, andphysiology of articular cartilage. Investig. Radiol. 35, 573–580




ww

.mcponline.org/

Dow

nloaded from


3. Dunlop, D. D., Manheim, L. M., Yelin, E. H., Song, J., and Chang, R. W.(2003) The costs of arthritis. Arthritis Rheum. 49, 101–113

4. Wieland, H. A., Michaelis, M., Kirschbaum, B. J., and Rudolphi, K. A. (2005)Osteoarthritis—an untreatable disease? Nat. Rev. Drug Discov. 4,331–344

5. Aigner, T. (2002) Towards a new understanding and classification of chon-drogenic neoplasias of the skeleton—biochemistry and cell biology ofchondrosarcoma and its variants. Virchows Arch. 441, 219–230

6. Rozeman, L. B., Hogendoorn, P. C., and Bovee, J. V. (2002) Diagnosis andprognosis of chondrosarcoma of bone. Expert Rev. Mol. Diagn. 2,461–472

7. Aigner, T., Zien, A., Gehrsitz, A., Gebhard, P. M., and McKenna, L. (2001)Anabolic and catabolic gene expression pattern analysis in normal ver-sus osteoarthritic cartilage using complementary DNA-array technology.Arthritis Rheum. 44, 2777–2789

8. Aigner, T., and Dudhia, J. (2003) Genomics of osteoarthritis. Curr. Opin.Rheumatol. 15, 634–640

9. Rozeman, L. B., Hameetman, L., van Wezel, T., Taminiau, A. H., Cleton-Jansen, A. M., Hogendoorn, P. C., and Bovee, J. V. (2005) cDNA ex-pression profiling of chondrosarcomas: Ollier disease resembles solitarytumours and alteration in genes coding for components of energy me-tabolism occurs with increasing grade. J. Pathol. 207, 61–71

10. Alaiya, A. A., Franzen, B., Auer, G., and Linder, S. (2000) Cancer proteom-ics: from identification of novel markers to creation of artificial learningmodels for tumor classification. Electrophoresis 21, 1210–1217

11. Lim, M. S., and Elenitoba-Johnson, K. S. (2004) Proteomics in pathologyresearch. Lab. Investig. 84, 1227–1244

12. Tilleman, K., Deforce, D., and Elewaut, D. (2005) Rheumatology: a closeencounter with proteomics. Rheumatology (Oxford) 44, 1217–1226

13. Hermansson, M., Sawaji, Y., Bolton, M., Alexander, S., Wallace, A., Begum,S., Wait, R., and Saklatvala, J. (2004) Proteomic analysis of articularcartilage shows increased type II collagen synthesis in osteoarthritis andexpression of inhibin �A (activin A), a regulatory molecule for chondro-cytes. J. Biol. Chem. 279, 43514–43521

14. Ruiz-Romero, C., Lopez-Armada, M. J., and Blanco, F. J. (2005) Proteomiccharacterization of human normal articular chondrocytes: a novel tool forthe study of osteoarthritis and other rheumatic diseases. Proteomics 5,3048–3059

15. Abbott, J., and Holtzer, H. (1966) The loss of phenotypic traits by differen-tiated cells. 3. The reversible behavior of chondrocytes in primary cul-tures. J. Cell Biol. 28, 473–487

16. Schnabel, M., Marlovits, S., Eckhoff, G., Fichtel, I., Gotzen, L., Vecsei, V.,and Schlegel, J. (2002) Dedifferentiation-associated changes in morphol-ogy and gene expression in primary human articular chondrocytes in cellculture. Osteoarthritis Cartilage 10, 62–70

17. Diaz-Romero, J., Gaillard, J. P., Grogan, S. P., Nesic, D., Trub, T., andMainil-Varlet, P. (2005) Immunophenotypic analysis of human articularchondrocytes: changes in surface markers associated with cell expan-sion in monolayer culture. J. Cell. Physiol. 202, 731–742

18. Wessel, D., and Flugge, U. I. (1984) A method for the quantitative recoveryof protein in dilute solution in the presence of detergents and lipids. Anal.Biochem. 138, 141–143

19. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal. Biochem. 72, 248–254

20. McKenna, L. A., Gehrsitz, A., Soder, S., Eger, W., Kirchner, T., and Aigner,T. (2000) Effective isolation of high-quality total RNA from human adultarticular cartilage. Anal. Biochem. 286, 80–85

21. Fessler, J. H., and Tandberg, W. D. (1975) Interactions between collagenchains and fiber formation. J. Supramol. Struct. 3, 17–23

22. Bovee, J. V., Cleton-Jansen, A. M., Taminiau, A. H., and Hogendoorn, P. C.(2005) Emerging pathways in the development of chondrosarcoma ofbone and implications for targeted treatment. Lancet Oncol. 6, 599–607

23. Choe, L. H., and Lee, K. H. (2003) Quantitative and qualitative measure ofintralaboratory two-dimensional protein gel reproducibility and the ef-fects of sample preparation, sample load, and image analysis. Electro-phoresis 24, 3500–3507

24. Schlags, W., Walther, M., Masree, M., Kratzel, M., Noe, C. R., and Lach-mann, B. (2005) Towards validating a method for two-dimensional elec-trophoresis/silver staining. Electrophoresis 26, 2461–2469

25. Rabilloud, T. (2000) Detecting proteins separated by 2-D gel electrophore-sis. Anal. Chem. 72, 48A–55A

26. Reed, G. F., Lynn, F., and Meade, B. D. (2002) Use of coefficient of variationin assessing variability of quantitative assays. Clin. Diagn. Lab. Immunol.9, 1235–1239

27. Nakagawa, K., Sakiyama, H., Fukazawa, T., Matsumoto, M., Takigawa, M.,Toyoguchi, T., and Moriya, H. (1997) Coordinated change between com-plement C1s production and chondrocyte differentiation in vitro. CellTissue Res. 289, 299–305

28. Sinz, A., Bantscheff, M., Mikkat, S., Ringel, B., Drynda, S., Kekow, J.,Thiesen, H. J., and Glocker, M. O. (2002) Mass spectrometric proteomeanalyses of synovial fluids and plasmas from patients suffering fromrheumatoid arthritis and comparison to reactive arthritis or osteoarthritis.Electrophoresis 23, 3445–3456

29. Ali, M., and Manolios, N. (2005) Proteomics in rheumatology: a new direc-tion for old diseases. Semin. Arthritis Rheum. 35, 67–76

30. Nakagawa, K., Sakiyama, H., Tsuchida, T., Yamaguchi, K., Toyoguchi, T.,Masuda, R., and Moriya, H. (1999) Complement C1s activation in de-generating articular cartilage of rheumatoid arthritis patients: immuno-histochemical studies with an active form specific antibody. Ann. Rheum.Dis. 58, 175–181

31. Sjoberg, A., Onnerfjord, P., Morgelin, M., Heinegard, D., and Blom, A. M.(2005) The extracellular matrix and inflammation: fibromodulin activatesthe classical pathway of complement by directly binding C1q. J. Biol.Chem. 280, 32301–32308

32. Steel, L. F., Trotter, M. G., Nakajima, P. B., Mattu, T. S., Gonye, G., andBlock, T. (2003) Efficient and specific removal of albumin from humanserum samples. Mol. Cell. Proteomics 2, 262–270




ww

.mcponline.org/

Dow

nloaded from


Documents

Establishment of a Reliable Method for Direct Proteome