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• W2105787434 abstract "The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration. The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration. Mesenchymal stem cells (MSCs) 1The abbreviations used are: MSCmesenchymal stem cell2Dtwo-dimensionalBMP-2bone morphogenetic protein 2ECMextracellular matrixFAformic acidFDRfalse discovery rateiTRAQisobaric tag for relative and absolute quantitationSCXstrong cation exchangeESembryonic stemLOXprotein-lysine 6-oxidaseMARCKSmyristoylated alanine-rich protein kinase C substrateHNRNPheterogeneous nuclear ribonucleoproteinnano-LCnanoscale LCIPIInternational Protein IndexsiRNAshort interfering RNAGAGglycosaminoglycanIGF-1Insulin-like growth factor-1PAPSS23′-phosphoadenosine 5′-phosphosulfate synthase 2MAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseR.S.D.relative S.D. are multipotent cells found in several adult tissues that can be expanded in vitro and differentiate into multiple mesoderm-type cells, including chondrocytes, thus representing a promising source for cell-based therapy of cartilage defects (1.Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17780) Google Scholar). Chondrogenic differentiation of MSCs in vitro closely resembles in vivo chondrogenesis, including mesenchymal cell condensation, chondrocyte differentiation, and maturation, which is elaborately modulated by signals initiated by cell-cell and cell-matrix interactions as well as a variety of growth and differentiation factors (2.DeLise A.M. Fischer L. Tuan R.S. Cellular interactions and signaling in cartilage development.Osteoarthritis Cartilage. 2000; 8: 309-334Abstract Full Text PDF PubMed Scopus (645) Google Scholar, 3.Kolf C.M. Cho E. Tuan R.S. Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation.Arthritis Res. Ther. 2007; 9: 204Crossref PubMed Scopus (727) Google Scholar, 4.Goldring M.B. Tsuchimochi K. Ijiri K. The control of chondrogenesis.J. Cell. Biochem. 2006; 97: 33-44Crossref PubMed Scopus (829) Google Scholar). Despite the enhanced interest and accumulating reports on making use of MSCs in cartilage repair and regeneration, the exact molecular events that occur in chondrogenic differentiation of MSCs remain largely unclear (5.Steinert A.F. Ghivizzani S.C. Rethwilm A. Tuan R.S. Evans C.H. Nöth U. Major biological obstacles for persistent cell-based regeneration of articular cartilage.Arthritis Res. Ther. 2007; 9: 213Crossref PubMed Scopus (243) Google Scholar).MSCs are routinely isolated from bone marrow according to their property of adhesion to plastic, which results in a morphologically, phenotypically, and functionally heterogeneous population of cells (6.Baksh D. Song L. Tuan R.S. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy.J. Cell. Mol. Med. 2004; 8: 301-316Crossref PubMed Scopus (893) Google Scholar, 7.Satomura K. Derubeis A.R. Fedarko N.S. Ibaraki-O'Connor K. Kuznetsov S.A. Rowe D.W. Young M.F. Gehron Robey P. Receptor tyrosine kinase expression in human bone marrow stromal cells.J. Cell. Physiol. 1998; 177: 426-438Crossref PubMed Scopus (92) Google Scholar). Because of the absence of defined markers, it is hard to obtain a morphologically and functionally homogeneous population, especially given the biological complexity derived from different ages and genetic backgrounds of the donors. To facilitate the molecular mechanism investigation and further in-depth biological function analysis, we chose a well established in vitro chondrogenic model in our primary proteomics study. C3H10T1/2, a murine embryonic mesenchymal cell line (8.Reznikoff C.A. Brankow D.W. Heidelberger C. Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division.Cancer Res. 1973; 33: 3231-3238PubMed Google Scholar), has been demonstrated to differentiate into multiple mesenchymal lineages such as chondrocytes, osteoblasts, and adipocytes (9.Shea C.M. Edgar C.M. Einhorn T.A. Gerstenfeld L.C. BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis.J. Cell. Biochem. 2003; 90: 1112-1127Crossref PubMed Scopus (172) Google Scholar). Therefore, this cell line is regarded as a model for MSCs, representing a homogeneous population of multipotential cells that do not spontaneously differentiate under normal culture conditions, and hence is an ideal vehicle for in vitro study of chondrogenesis (10.Denker A.E. Haas A.R. Nicoll S.B. Tuan R.S. Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. stimulation by bone morphogenetic protein-2 in high-density micromass cultures.Differentiation. 1999; 64: 67-76Crossref PubMed Scopus (215) Google Scholar). Additionally, a recent report by Zhao et al. (11.Zhao L. Li G. Chan K.M. Wang Y. Tang P.F. Comparison of multipotent differentiation potentials of murine primary bone marrow stromal cells and mesenchymal stem cell line C3H10T1/2.Calcif. Tissue Int. 2009; 84: 56-64Crossref PubMed Scopus (50) Google Scholar) revealed that under the same inductive conditions C3H10T1/2 cells showed chondrogenic differentiation potentials comparable to primary murine bone marrow-derived MSCs, suggesting that this cell line is a good alternative cell source for investigating chondrogenic differentiation. Consistent with a previous report (10.Denker A.E. Haas A.R. Nicoll S.B. Tuan R.S. Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. stimulation by bone morphogenetic protein-2 in high-density micromass cultures.Differentiation. 1999; 64: 67-76Crossref PubMed Scopus (215) Google Scholar), chondrogenic differentiation of C3H10T1/2 cells was induced by a high density micromass environment and BMP-2 treatment in the present study. This in vitro model has also been used in gene expression profiling of mesenchymal chondrogenesis (12.Izzo M.W. Pucci B. Tuan R.S. Hall D.J. Gene expression profiling following BMP-2 induction of mesenchymal chondrogenesis in vitro.Osteoarthritis Cartilage. 2002; 10: 23-33Abstract Full Text PDF PubMed Scopus (19) Google Scholar).Recently, proteomics approaches have been applied to the studies of MSCs, such as the secretome of embryonic stem (ES) cell-derived MSCs (13.Sze S.K. de Kleijn D.P. Lai R.C. Khia Way Tan E. Zhao H. Yeo K.S. Low T.Y. Lian Q. Lee C.N. Mitchell W. El Oakley R.M. Lim S.K. Elucidating the secretion proteome of human embryonic stem cell-derived mesenchymal stem cells.Mol. Cell. Proteomics. 2007; 6: 1680-1689Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), the global effects of transforming growth factor-β on MSCs (14.Wang D. Park J.S. Chu J.S. Krakowski A. Luo K. Chen D.J. Li S. Proteomic profiling of bone marrow mesenchymal stem cells upon transforming growth factor beta1 stimulation.J. Biol. Chem. 2004; 279: 43725-43734Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), differential expression profiling of membrane proteins of MSCs undergoing osteoblast differentiation (15.Foster L.J. Zeemann P.A. Li C. Mann M. Jensen O.N. Kassem M. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation.Stem Cells. 2005; 23: 1367-1377Crossref PubMed Scopus (186) Google Scholar), and proteome analysis of rat MSC subcultures (16.Celebi B. Elçin Y.M. Proteome analysis of rat bone marrow mesenchymal stem cell subcultures.J. Proteome Res. 2009; 8: 2164-2172Crossref PubMed Scopus (30) Google Scholar). However, the comprehensive expression profiling of MSCs undergoing chondrogenic differentiation has not been reported yet (17.Park H.W. Shin J.S. Kim C.W. Proteome of mesenchymal stem cells.Proteomics. 2007; 7: 2881-2894Crossref PubMed Scopus (64) Google Scholar). To gain further understanding of the molecular mechanism underlying this stringently modulated process, we applied an iTRAQ labeling coupled with on-line 2D LC/MS/MS proteomics technology to quantitatively assess the protein expression profile of the in vitro chondrogenesis model. The iTRAQ labeling coupled with LC/MS/MS is a gel-free quantitative proteomics technology that uses amine-specific isobaric tags to compare the intensity of reporter ions of labeled peptides and infer quantitative values for corresponding proteins (18.Wiese S. Reidegeld K.A. Meyer H.E. Warscheid B. Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research.Proteomics. 2007; 7: 340-350Crossref PubMed Scopus (579) Google Scholar).In this study, we compared the protein profile of chondrogenically differentiated C3H10T1/2 cells with those of undifferentiated cells by using iTRAQ labeling coupled with on-line 2D LC/MS/MS. A total of 1756 proteins were identified. Of them, 100 significantly altered proteins were identified (95% confidence interval), and six of them were validated by Western blotting. Further functional categorization revealed that these altered proteins could play essential roles in lineage-specific differentiation, promoting a nonspecific stem cell state or the commitment to other lineages. Finally, biological functions of a number of these intriguing proteins were preliminarily validated in the context of chondrogenesis. Such findings will advance our understanding of the underlying intracellular mechanisms that modulate chondrogenic differentiation as well as self-renewal of MSCs and in turn promote their application in cartilage defect therapy.EXPERIMENTAL PROCEDURESCell CultureCell culture media and media supplements were purchased from Invitrogen. C3H10T1/2 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). Monolayer cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 50 units/ml penicillin G, and 50 μg/ml streptomycin and incubated at 37 °C in 5% (v/v) CO2 in a humidified incubator. Media were changed every 3 days.Chondrogenesis InductionThe micromass culture technique modified from Ahrens et al. (19.Ahrens P.B. Solursh M. Reiter R.S. Stage-related capacity for limb chondrogenesis in cell culture.Dev. Biol. 1977; 60: 69-82Crossref PubMed Scopus (537) Google Scholar) was adopted for chondrogenic differentiation experiments. Briefly, subconfluent C3H10T1/2 cells were trypsinized and resuspended at a concentration of 107 cells/ml in Ham's F-12 medium with 10% (v/v) fetal bovine serum. A 10-μl drop of cell suspension was placed in a well of a 24-well culture plate and allowed to adhere for 2–3 h at 37 °C and 5% (v/v) CO2, then 1 ml of medium containing 100 ng/ml human recombinant BMP-2 purchased from R&D Systems (Minneapolis, MN) was added to the cultures, and controls were cultured in the same manner but without supplementation of human recombinant BMP-2. The cultures were fed every 3 days with fresh media with or without BMP-2, and day 6 cultures were used for the proteomics analysis.Alcian Blue Staining and QuantificationFor Alcian blue staining, cultures were rinsed twice with PBS, fixed in 4% (w/v) paraformaldehyde for 15 min, and incubated in 1% (w/v) Alcian blue 8-GX (Sigma) in 0.1 n HCl (pH 1.0) overnight. For quantitative analysis, Alcian blue-stained cultures were extracted with 6 m guanidine HCl for 2 h at room temperature. The absorption of the extracted dye was measured at 650 nm in a microplate reader (Bio-Rad).Western BlottingCultures were washed with ice-cold phosphate buffered saline and lysed in a buffer containing 50 mm Tris-HCl (pH 7.6), 5 mm EDTA, 50 mm NaCl, 30 mm sodium pyrophosphate, 50 mm NaF, 0.1 mm Na3VO4, 1% (v/v) Triton X-100, 1 mm PMSF, and a protease inhibitor mixture tablet (Roche Applied Science). Lysates were clarified by centrifugation at 15,000 × g for 10 min at 4 °C, and the protein concentration of the supernatant was measured by the Bradford protein assay (Bio-Rad). Samples containing 30 μg of total protein were separated by 12% (w/v) SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA). After incubating for 1 h with blocking buffer (5% (w/v) nonfat milk in TBS-T (0.05% (v/v) Tween 20 in Tris-buffered saline)), the membrane was probed with the indicated primary antibodies diluted in blocking buffer overnight at 4 °C. After being extensively washed with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated antibody to mouse (Kirkegaard and Perry Laboratories, Inc.) or rabbit (Cell Signaling Technology) diluted in blocking buffer (1:2000) for 1 h at room temperature. Bands were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and recorded on x-ray films (Fuji Medical, Tokyo, Japan). Finally, the visualized bands were quantified by QUANTITY ONE software on a GS-800 densitometer (Bio-Rad). The following antibodies were used: monoclonal anti-collagen type II (Lab Vision Corp.) (1:1000), monoclonal anti-β-tubulin (Sigma-Aldrich) (1:5000), rabbit polyclonal antibody to protein-lysine 6-oxidase (LOX) (Santa Cruz Biotechnology, Inc.) (1:500), rabbit polyclonal antibody to Histone H2A.X (Abcam) (1:2000), and monoclonal antibodies to MARCKS and hnRNPM (Santa Cruz Biotechnology, Inc.) (1:1000).Flow CytometryFor flow cytometric analysis, the cultures were detached by 0.1% (w/v) trypsin and collagenase type II (Sigma-Aldrich) digestion followed by washing and fixation. The resulting pellets were resuspended in 1% (w/v) bovine serum albumin (Roche Applied Science) for 30 min in room temperature to block the nonspecific binding sites. Afterward, the samples were incubated with anti-collagen type II monoclonal antibody (Clone 2B1.5, Neomarker) at 4 °C for 8 h and then stained with a FITC-conjugated goat anti-mouse secondary antibody (Molecular Probes, Inc.) at room temperature for 1 h. Flow cytometric acquisition and data analysis were performed with an Epics ALTRA flow cytometer and EXPO32TM software (Beckman Coulter, Inc., Miami, FL). As a negative control, the cells were incubated only with the FITC-conjugated secondary antibody. Three independent flow cytometric experiments were performed.iTRAQ Labeling, Sample Cleaning, and DesaltingAll the reagents and buffers needed for iTRAQ labeling and cleaning were obtained from Applied Biosystems (Foster City, CA). iTRAQ labeling was performed according to the manufacturer's instructions. Briefly, at day 6, total cell lysates were collected as described for Western blotting, and protein concentration was determined by the Bradford assay. Subsequently, 100 μg of protein was precipitated with ice-cold acetone overnight at −20 °C. Protein pellets were dissolved, denatured, alkylated, and digested with trypsin (Sigma; 1:20, w/w, 37 °C for 18 h). To label peptides with iTRAQ reagent, 1 unit of label (defined as the amount of reagent required to label 100 μg of protein) was thawed and reconstituted in 70 μl of ethanol; digestions from BMP-2-treated and untreated C3H10T1/2 cells were labeled with 117and 114 iTRAQ reagents, respectively; and then the samples were incubated at room temperature for 1 h and pooled. In the present study, three independent chondrogenic induction and iTRAQ labeling experiments were carried out.Prior to on-line 2D nanoscale LC (nano-LC)/MS/MS analysis, iTRAQ-labeled samples were cleaned up and desalted. A cation exchange cartridge system (Applied Biosystems) was used to remove the reducing reagent, SDS, excess iTRAQ reagents, undigested proteins, and trypsin in the labeled sample mixture that would interfere with the LC/MS/MS analysis. Before cation exchange, the concentration of buffer salts in the labeled samples was reduced below 10 mm by diluting 10-fold with cation exchange buffer-load (10 mm K2HPO4 in 25% (v/v) acetonitrile at pH 3.0). The pH of the diluted sample was checked: it should be between 2.5 and 3.3; otherwise it was adjusted to 3.0 with phosphoric acid. The diluted sample mixture was loaded onto the cation exchange cartridge, after washing with 10 column volumes of buffer-load, peptides were eluted with 500 μl of buffer-elute (10 mm K2HPO4 in 25% (v/v) acetonitrile, 350 mm KCl at pH 3.0). Afterward, the eluate of the cation exchange was desalted using an Agilent 1100 series HPLC system equipped with an autosampler, 2/6 valve, and diode array detector (220 nm) (Agilent, Waldbronn, Germany). Eluates of cation exchange were diluted with 0.1% (v/v) FA (v/v in H2O) to reduce the concentration of acetonitrile to 5% (v/v), afterward the dilution was injected onto a 4.6-mm-inner diameter × 150-mm C18 reversed-phase column (5 μm, 80 Å; Agilent, Waldbronn, Germany), flushed with phase A (5% (v/v) acetonitrile, 0.1% (v/v) FA in H2O) at 1 ml/min for 10 min, and finally peptides were eluted with 65% (v/v) phase B (90% (v/v) acetonitrile, 0.1% (v/v) FA in H2O). Absorbance at 220 nm was monitored, and the maximal absorption peak was collected and divided into two aliquots. Each aliquot was dried in a Heto vacuum concentrator (Heto-Holten A/S, Allerod, Denmark).On-line 2D LC/MS/MS2D nano-LC/MS/MS analyses were conducted on a nano-HPLC system (Agilent, Waldbronn, Germany) coupled to a hybrid Q-TOF mass spectrometer (QSTAR XL, Applied Biosystems) equipped with a nano-ESI source (Applied Biosystems) and a nano-ESI needle (Picotip, FS360-50-20; New Objective Inc., Woburn, MA). AnalystTM 1.1 software was used to control QSTAR XL mass spectrometry and the nano-HPLC system and to acquire mass spectra. Vacuum-dried iTRAQ-labeled peptides were reconstituted in phase A and injected at a flow rate of 10 μl/min onto a high resolution strong cation exchange (SCX) column (Bio-SCX, 300-μm inner diameter × 35 mm; Agilent, Wilmington, DE), which was on line with a C18 precolumn (PepMap, 300-μm inner diameter × 5 mm; LC Packings). After loading, the SCX column and C18 precolumn were flushed with a 16-step gradient sodium chloride solution (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 300, and 400 mm) for 5 min and phase A for 10 min at a flow rate of 15 μl/min. Afterward, the precolumn was switched on line with a nanoflow reversed-phase column (VYDAC 218MS, 75-μm inner diameter × 100 mm; Grace, Hesperia, CA), and the peptides concentrated and desalted on the precolumn were separated using a 120-min linear gradient from 12 to 30% (v/v) phase B (0.1% (v/v) FA in ACN) at a flow rate of 400 nl/min.The Q-TOF instrument was operated in positive ion mode with ion spray voltage typically maintained at 2.0 kV. Mass spectra of iTRAQ-labeled samples were acquired in an information-dependent acquisition mode. The analytical cycle consisted of a 0.7-s MS survey scan (400–1600 m/z) followed by three 2-s MS/MS scans (100–2000 m/z) of the three most abundant peaks (i.e. precursor ions), which were selected from the MS survey scan. Precursor ion selection was based upon ion intensity (peptide signal intensity above 25 counts/s) and charge state (2+ to 5+), and once the ions were fragmented in the MS/MS scan, they were allowed one repetition before a dynamic exclusion for a period of 120 s. Because of the iTRAQ tags, the parameters for rolling collision energy (automatically set according to the precursor m/z and charge state) were manually optimized. Under CID, iTRAQ-labeled peptides fragmented to produce reporter ions at 114.1 and 117.1, and fragment ions of the peptides were simultaneously produced, resulting in sequencing of the labeled peptides and identification of the corresponding proteins. The ratios of the peak areas of the two iTRAQ reporter ions reflected the relative abundances of the peptides and the proteins in the samples. External calibration of mass spectrometer was carried out using reserpine and trypsinized BSA routinely.Protein Identification and False Discovery Rate (FDR) AnalysisFor protein identification, the complete set of raw data files (*.wiff) of each run was analyzed together using ProteinPilot software 2.0.1 (revision 67476), which consisted of the Paragon and Pro GroupTM algorithms. The Paragon search algorithm used a sequence tag algorithm to calculate sequence temperature values for identification of peptides from a database (20.Shilov I.V. Seymour S.L. Patel A.A. Loboda A. Tang W.H. Keating S.P. Hunter C.L. Nuwaysir L.M. Schaeffer D.A. The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra.Mol. Cell. Proteomics. 2007; 6: 1638-1655Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar). The parameters for searching were as follows: iTRAQ fourplex peptide labeled; trypsin digestion; methyl methane thiosulfate alkylation of cysteine residue; instrument, QSTAR ESI; identification focus, biological modifications; non-redundant International Protein Index (IPI) mouse sequence database version 3.62 (date of release, August 2009; with a total of 56,727 protein entries) from the European Bioinformatics Institute selected; and software defaults used for other parameters needed for searching.Afterward, the Pro Group algorithm assembled the peptide evidence from the Paragon algorithm to find the smallest number of proteins that could explain all the fragmentation spectral evidence. The core philosophy of the Pro Group algorithm was that the spectral evidence used to prove the detection of one protein could not be used again to prove the detection of a second protein; therefore, two types of scores for each protein were calculated: Total ProtScore and Unused ProtScore. The Total ProtScore was based on all peptides pointing to a protein and was analogous to protein scores reported by other protein identification softwares, whereas the Unused ProtScore was calculated from the peptide evidence that had not already been used to explain more confident proteins. The Pro Group algorithm greatly reduced the redundancy and suppressed the false positives. In addition, the Pro Group algorithm also could distinguish protein isoforms; a specific protein isoform would be reported only if unique evidence (peptide) existed for this isoform (from Understanding the Pro Group Algorithm, Applied Biosystems). To obtain an even more reliable protein list, in the present study, proteins were identified on the basis of having at least two distinct peptides with 99% confidence and 2.0 contribution to Unused ProtScore. In other words, only proteins with Unused ProtScore greater than or equal to 4.0 were included in the final protein list.To evaluate the rate of erroneously identified proteins, the same set of MS spectral data was searched as above but against a decoy database, a randomized version of the database generated by DecoyDBB software (21.Reidegeld K.A. Eisenacher M. Kohl M. Chamrad D. Körting G. Blöggel M. Meyer H.E. Stephan C. An easy-to-use Decoy Database Builder software tool, implementing different decoy strategies for false discovery rate calculation in automated MS/MS protein identifications.Proteomics. 2008; 8: 1129-1137Crossref PubMed Scopus (68) Google Scholar). The following formula was used to calculate the FDR of protein identification: FDR = (FP/FP + TP) × 100% (FP, false positives, number of random hits; TP, true positives, number of normal hits).Protein Quantification and Identification of Differentially Expressed ProteinsProtein quantification was also performed by ProteinPilot software, which automatically calculated the relative abundance of iTRAQ-labeled peptides and the corresponding proteins. Corrections were made for impurity of iTRAQ reagents based on the data provided by the manufacturer. For pipetting and other similar errors in analyses, iTRAQ ratios were normalized by autobias, which used all data obtained from a 2D LC/MS/MS analysis to calculate the bias correction factor.In the present study, the thresholds for protein differential expression were mean ± 1.96S.D. (95% confidence interval). Proteins whose 117/114 ratio met the differential expression threshold in at least two of the three biological experiments are shown in the main text, whereas those that appeared in only one of the three biological experiments are listed in the supplemental material.RNA Extraction and Quantitative Real Time PCRThe methods for RNA extraction and quantitative real time PCR were the same as in our previous report (22.Pan Q. Yu Y. Chen Q. Li C. Wu H. Wan Y. Ma J. Sun F. Sox9, a key transcription factor of bone morphogenetic protein-2-induced chondrogenesis, is activated through BMP pathway and a CCAAT box in the proximal promoter.J. Cell. Physiol. 2008; 217: 228-241Crossref PubMed Scopus (110) Google Scholar). Briefly, total RNA was extracted and purified using the RNeasy kit (Qiagen, Valencia, CA). Gene products were analyzed by using SYBR Green PCR Master Mix (Applied Biosystems) and specific primers in a 7300 Real Time PCR System (Applied Biosystems). Relative gene expression levels were calculated as ratios of the mRNA levels normalized against those of 18 S rRNA. All the results were expressed as means ± S.D. of three independent experiments. Primer sequences are reported in supplemental Table 1.Lactate AssayLactate concentration in the conditioned medium of day 6 cultures was measured by using a lactate assay kit (Biovision) according to the manufacture's instructions. The absorption at 570 nm was measured by a microplate reader (Bio-Rad).Short Interfering RNA (siRNA) TransfectionON-TARGETplus SMARTpool siRNA against Btf3l4 (L-045446-01-0005) or Fbln5 (M-059161-01-0005) was transfected into the C3H10T1/2 cells, respec" @default.
• W2105787434 created "2016-06-24" @default.
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• W2105787434 date "2010-03-01" @default.
• W2105787434 modified "2023-10-13" @default.
• W2105787434 title "Quantitative Proteomics Analysis of Chondrogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells by iTRAQ Labeling Coupled with On-line Two-dimensional LC/MS/MS" @default.
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