Objective: To adopt liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze surface marker proteins of mesenchymal stem cells (MSCs), exploring a novel methodology for their identification. Methods: MSCs were lysed with RIPA buffer, and the protein concentration of cell lysates was quantified using a BCA kit. After reduction and alkylation of MSCs proteins, trypsin digestion was performed, and the resulting peptides were analyzed by LC-MS/MS. The chromatographic separation was carried out on An ACQUITY Premier Peptide BEH C18 column (150 mm×2.1 mm, 1.7 µm) with the mobile phase consisting of water containing 0.1% formic acid(A)-acetonitrile containing 0.1% formic acid(B) in a gradient mode at a flow rate of 0.2 mL · min-1. The mass spectrometer was operated in positive ion mode, sensitivity, data-dependent acquisition (DDA) mode, with capillary voltage set at 2.5 kV, cone voltage at 40 V, ion source temperature at 120 ℃, and a scan range of m/z 50-2 000. Proteins were identified by database searching using waters connect software. Mass tolerance was set as 0.001% for precursor ions and 0.002% for fragmentation ions. And a minimum of three b/y-ion matches were set as the criteria for peptide identification. Results: Through DDA mode, the positive marker CD90 of MSCs was identified. Further introduction of an include list under DDA mode led to the identification of the positive marker CD73, while the positive marker CD105 was not identified. Negative markers including CD34, CD45, CD14, CD19, CD79a, and HLA-DR were not detected. Conclusion: LC-MS/MS technology is rapid, accurate, and antibody-independent. It has identified two positive marker proteins. Through further research and methodological exploration, this technique can be applied to the identification of MSC surface marker proteins and target proteins.
[1] SALEHINEJAD P,MOSHREFI M,ESLAMINEJAD T.An overview on mesenchymal stem cells derived from extraembryonic tissues:supplement sources and isolation methods[J].Stem Cells Cloning,2020,13:57
[2] WATANABE Y,TSUCHIYA A,TERAI,S.The development of mesenchymal stem cell therapy in the present,and the perspective of cell-free therapy in the future[J].Clin Mol Hepatol,2021,27(1):70
[3] DING DC,SHYU WC,LIN SZ.Mesenchymal stem cells[J].Cell transplant,2011,20(1):5
[4] 黄贵才,罗业浩,江慧容,等.干细胞对肝脏疾病的临床治疗前景与研究现状[J].中国组织工程研究,2023,27(19):3091
HUANG GC,LUO YH,JIANG HR,et al.Clinical therapeutic prospect and current research status of stem cells for liver diseases[J].Chin J Tissue Eng Res,2023,27(19):3091
[5] 毛开云,范月蕾,王跃,等.间充质干细胞治疗产品开发现状与趋势[J].中国生物工程杂志,2017,37(10):126
MAO KY,FAN YL,WANG Y,et al.Development status and trend analysis of mesenchymal stem cells therapeutic products[J].China Biotechnol,2017,37(10):126
[6] NAJI A,EITOKU M,FAVIER B,et al.Biological functions of mesenchymal stem cells and clinical implications[J].Cell Mol Life Sci,2019,76:3323
[7] DOMINICI M,BLANC K,MUELLER I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement[J].Cytotherapy,2006,8(4):315
[8] MALEKI M,GHANBARVAND F,BEHVARZ MR,et al.Comparison of mesenchymal stem cell markers in multiple human adult stem cells[J].Int J Stem Cells,2014,7(2):118
[9] HODGKINSON CP,GOMEZ JA,MIROTSOU M,et al.Genetic engineering of mesenchymal stem cells and its application in human disease therapy[J].Hum Gene Ther,2010,21:1513
[10] NAKAMURA K,ITO Y,KAWANO Y,et al.Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model[J].Gene Ther,2004,11:1155
[11] YANG D,SUN S,WANG Z,et al.Stromal cell-derived factor-1 receptor CXCR4-overexpressing bone marrow mesenchymal stem cells accelerate wound healing by migrating into skin injury areas[J].Cell Reprogram,2013,15:206
[12] AMARA I,TOUATI W,BEAUNE P,et al.Mesenchymal stem cells as cellular vehicles for prodrug gene therapy against tumors[J].Biochimie,2014,105:4
[13] MUELLER LP,LUETZKENDORF J,WIDDER M,et al.TRAIL-transduced multipotent mesenchymal stromal cells(TRAIL-MSC)overcome TRAIL resistance in selected CRC cell lines in vitro and in vivo[J].Cancer Gene Ther,2011,18:229
[14] CHOI S,HONG JA,CHOI HJ,et al.Enhanced tumor targeting and timely viral release of mesenchymal stem cells/oncolytic virus complex due to GRP78 and inducible E1B55K expressions greatly increase the antitumor effect of systemic treatment[J].Mol Ther Oncolytics,2022,27:26
[15] 康赵飞,王可心,韩春乐,等.液质联用鉴定重组CHO细胞株目的蛋白的表达研究[J].药物分析杂志,2025,45(1):99
KANG ZF,WANG KX,HAN CL,et al.Study of liquid chromatography-mass spectrometry identification of target protein expression in recombinant CHO cell lines[J].Chin J Pharm Anal,2025,45(1):99
[16] MATTA C,BOOCOCK DJ,FELLOWS CR,et al.Molecular phenotyping of the surfaceome of migratory chondroprogenitors and mesenchymal stem cells using biotinylation,glycocapture and quantitative LC-MS/MS proteomic analysis[J].Sci Rep,2019,9(1):9018
[17] MANUGUERRA GR,BOULOS PR,AMMAR A,et al.Transplantation of mesenchymal stem cells promotes tissue regeneration in a glaucoma model through laser-induced paracrine factor secretion and progenitor cell recruitment[J].Stem Cells,2013,31:1136
[18] YANG F,LEUNG VY,LUK KD,et al.Mesenchymal stem cells arrest intervertebral disc degeneration through chondrocytic differentiation and stimulation of endogenous cells[J].Mol Ther,2009,17:1959
[19] NOTH U,STEINERT AF,TUAN RS.Technology insight:adult mesenchymal stem cells for osteoarthritis therapy[J].Nat Clin Pract Rheumatol,2008,4:371
[20] NIEHAGE C,STEENBLOCK C,PURSCHE T,et al.The cell surface proteome of human mesenchymal stromal cells[J].PLoS One,2011,6(5):e20399
[21] DONNENBERG AD,MEYER EM,RUBIN JP,et al.The cell-surface proteome of cultured adipose stromal cells[J].Cytometry A,2015,87(7):665
[22] WALMSLEY GG,ATASHROO DA,MAAN ZN,et al.High-throughput screening of surface marker expression on undifferentiated and differentiated human adipose-derived stromal cells[J].Tissue Eng Part A,2015,21(15-16):2281
[23] BAER PC,KUCI S,KRAUSE M,et al.Comprehensive phenotypic characterization of human adipose-derived stromal/stem cells and their subsets by a high throughput technology[J].Stem Cells Dev,2013,22(2):330
[24] CAMILLERI ET,GUSTAFSON MP,DUDAKOVIC A,et al.Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production[J].Stem Cell Res Ther,2016,7(1):107
[25] WHITELEGGE JP.Integral membrane proteins and bilayer proteomics[J].Anal Chem,2013,85(5):2558
