An Introduction to Analytical Challenges, Approaches, and Applications in Mass Spectrometry–Based Secretomics

Abstract

•Overview of challenges, approaches, and opportunities of LC-MS–based secretomics.•Guidance for unbiased analysis of secreted proteins from cultured cells.•Strength and weaknesses of different secretomics approaches. The active release of proteins into the extracellular space and the proteolytic cleavage of cell surface proteins are key processes that coordinate and fine-tune a multitude of physiological functions. The entirety of proteins that fulfill these extracellular tasks are referred to as the secretome and are of special interest for the investigation of biomarkers of disease states and physiological processes related to cell-cell communication. LC-MS–based proteomics approaches are a valuable tool for the comprehensive and unbiased characterization of this important subproteome. This review discusses procedures, opportunities, and limitations of mass spectrometry–based secretomics to better understand and navigate the complex analytical landscape for studying protein secretion in biomedical science. The active release of proteins into the extracellular space and the proteolytic cleavage of cell surface proteins are key processes that coordinate and fine-tune a multitude of physiological functions. The entirety of proteins that fulfill these extracellular tasks are referred to as the secretome and are of special interest for the investigation of biomarkers of disease states and physiological processes related to cell-cell communication. LC-MS–based proteomics approaches are a valuable tool for the comprehensive and unbiased characterization of this important subproteome. This review discusses procedures, opportunities, and limitations of mass spectrometry–based secretomics to better understand and navigate the complex analytical landscape for studying protein secretion in biomedical science. Multicellular organisms depend on the dynamic interplay of different organs, tissues, and cell types to sense and respond adequately to changes in the environment. The organization and orchestration of such responses is dependent on an efficient communication of signals between cells. On the molecular level, proteins play a major role as signaling cues for the transmission and reception of signals and can herby act either close by or in far distance. As such, an important class of proteins are those that are either actively released by a cell into the extracellular environment or reach the extracellular milieu via tissue leakage. The entirety of these proteins in the extracellular space are designated as the secretome (1Uhlen M. Karlsson M.J. Hober A. Svensson A.S. Scheffel J. Kotol D. et al.The human secretome.Sci. Signal. 2019; 12eaaz0274Google Scholar). Functionally, secreted proteins make up a diverse group of proteins covering growth factors, extracellular matrix constituents, cytokines, or hormones. According to the UniProtKB (accessed December 2022, keyword: secreted), 2097 of 20,401 reviewed proteins in total are annotated as secreted, suggesting that approximately 10% of the human proteome are potentially released via the classical secretory pathway or unconventional secretion processes. However, a growing number of experimental data have shown that protein secretion can be uncoupled from the classical endoplasmic reticulum (ER)-Golgi pathway, suggesting that unconventional protein secretion is an important factor which contributes to the active release of proteins into the extracellular space under certain conditions (2Villarreal L. Mendez O. Salvans C. Gregori J. Baselga J. Villanueva J. Unconventional secretion is a major contributor of cancer cell line secretomes.Mol. Cell Proteomics. 2013; 12: 1046-1060Google Scholar, 3Zhang M. Liu L. Lin X. Wang Y. Li Y. Guo Q. et al.A translocation pathway for vesicle-mediated unconventional protein secretion.Cell. 2020; 181: 637-652.e615Google Scholar, 4Phulphagar K. Kuhn L.I. Ebner S. Frauenstein A. Swietlik J.J. Rieckmann J. et al.Proteomics reveals distinct mechanisms regulating the release of cytokines and alarmins during pyroptosis.Cell Rep. 2021; 34108826Google Scholar, 5Meissner F. Scheltema R.A. Mollenkopf H.J. Mann M. Direct proteomic quantification of the secretome of activated immune cells.Science. 2013; 340: 475-478Google Scholar, 6Dong L.F. Kovarova J. Bajzikova M. Bezawork-Geleta A. Svec D. Endaya B. et al.Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells.Elife. 2017; 6e22187Google Scholar, 7Hurwitz S.N. Rider M.A. Bundy J.L. Liu X. Singh R.K. 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Biol. 2015; 427: 1202-1210Google Scholar). Secreted proteins are in the center of modified signaling pathways of numerous diseases, such as cancer (12Planque C. Kulasingam V. Smith C.R. Reckamp K. Goodglick L. Diamandis E.P. Identification of five candidate lung cancer biomarkers by proteomics analysis of conditioned media of four lung cancer cell lines.Mol. Cell Proteomics. 2009; 8: 2746-2758Google Scholar, 13Makridakis M. Vlahou A. Secretome proteomics for discovery of cancer biomarkers.J. Proteomics. 2010; 73: 2291-2305Google Scholar), cardiovascular (14Ranganath S.H. Levy O. Inamdar M.S. Karp J.M. Harnessing the mesenchymal stem cell secretome for the treatment of cardiovascular disease.Cell Stem Cell. 2012; 10: 244-258Google Scholar), neurodegenerative (15Carvalho M.M. Teixeira F.G. Reis R.L. Sousa N. Salgado A.J. Mesenchymal stem cells in the umbilical cord: phenotypic characterization, secretome and applications in central nervous system regenerative medicine.Curr. Stem Cell Res. Ther. 2011; 6: 221-228Google Scholar), and chronic liver diseases (16Kim K. Kim K.H. Targeting of secretory proteins as a therapeutic strategy for treatment of Nonalcoholic Steatohepatitis (NASH).Int. J. Mol. Sci. 2020; 21: 2296Google Scholar) or obesity (17Pardo M. Roca-Rivada A. Seoane L.M. Casanueva F.F. Obesidomics: contribution of adipose tissue secretome analysis to obesity 2012; Scholar) and important for and to disease or responses (1Uhlen M. Karlsson M.J. Hober A. Svensson A.S. Scheffel J. Kotol D. et al.The human secretome.Sci. Signal. 2019; 12eaaz0274Google Scholar). The investigation of proteins that are actively and released by cells is the of this a of and of LC-MS–based the that are the analysis of secreted and of biomedical LC-MS–based proteomics to be a key analytical tool for biomedical and for the investigation of signaling or for the of and However, a investigation of secreted proteins and signaling by LC-MS–based proteomics of and LC-MS–based proteomics is a valuable tool for the characterization and analysis of as to an unbiased and comprehensive of the entirety of secreted The to of proteins and data LC-MS–based secretomics approaches from that the analysis of a of proteins and of the proteins in the investigation of secreted proteins by mass a of the and limitations which are secretome analysis in and the analysis by mass in an of the and that to be for the analysis of major of secretomics is the of secreted proteins from the number of potentially the of the analysis and the of from a experimental that The experimental be on a treatment to and to be to the of cell of to which proteins released a and which are secreted or have into the The experimental a on the and of the cell such as cells are secretory active S. M. secretomics in Cell Proteomics. Scholar), the of secretomics in a and the of a multitude of different proteins However, the and quantification of cytokines, for which are in of immune cells to to the for the and the of quantification across different experimental conditions or different to a or is important for secretomics The of proteomics approaches which are on the and and as reviewed in M. S. M.M. B. mass in review from to the 2012; Scholar, S. K. M. C. K. mass an Mol. Biol. 2021; Scholar). protein mass are in a in which the are during the for the and on the cell type and the of the secretomics of proteins molecular and such as secreted cytokines, in in the of in a in the that prevents the for a and approaches S. et the of proteome profiling and to liver Cell Proteomics. 2015; Scholar, S. N. L. et data of the by a for and proteome Cell Proteomics. 2012; Scholar) such as C. L. G. S. for a Biol. Scholar) are and have in secretomics J. Muller J. L. M. et proteomics the cell J. 2020; Scholar). a are to proteome and common of secretomics is the of of a of proteins are annotated as secreted or extracellular F. Scheltema R.A. Mollenkopf H.J. Mann M. Direct proteomic quantification of the secretome of activated immune cells.Science. 2013; 340: 475-478Google Scholar, A.S. J. F. Mann M. Secretome analysis of in cells by a experimental and Res. 2015; Scholar). under the the cell have a and a number of cells or which to the and for can this or cell cell is for secretomics and the contribution of cell and to the secretome be of the can be quantification of release into the cell or of cells A.S. J. F. Mann M. Secretome analysis of in cells by a experimental and Res. 2015; Scholar). to the contribution of proteins to the mass in the secretome such as proteins or as an (2Villarreal L. Mendez O. Salvans C. Gregori J. Baselga J. Villanueva J. Unconventional secretion is a major contributor of cancer cell line secretomes.Mol. Cell Proteomics. 2013; 12: 1046-1060Google Scholar, O. Villanueva J. and opportunities for cell line in cancer 2015; Scholar, F. O. protein and an on cancer cell Res. 2006; Scholar). of proteins can be an for the of the secretome to processes or cell The of secretome are in R.L. S. et in the proteomic investigation of the cell Proteomics. 2012; Scholar, Y. to the of the cell Proteomics. Scholar) cell cells on the of or to a environment for cell growth and functions. However, the of or media a major as to a dynamic of protein of that to be by the mass such as of up to in the the of secreted proteins that in a of M. A. C. a better analysis of secreted the of the cells 7: Scholar, S. to the cell 2013; Scholar). the dynamic the for secretomics analysis is cell F. Scheltema R.A. Mollenkopf H.J. Mann M. Direct proteomic quantification of the secretome of activated immune cells.Science. 2013; 340: 475-478Google Scholar, A.S. J. F. Mann M. 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However, of for the experimental is the of for or which to secretion from secretome from cells to the cell and and as such, the secretome by of the between the and the The proteins are to a proteomics The of the secretome can be different approaches, such as protein or However, the of on the experimental and the such as classical or for for and cells for the investigation of secretion M. A. C. a better analysis of secreted the of the cells 7: Scholar). and have for secretome of of liver cell and F. Scheltema R.A. Mollenkopf H.J. Mann M. Direct proteomic quantification of the secretome of activated immune cells.Science. 2013; 340: 475-478Google Scholar, S. M. secretomics in Cell Proteomics. Scholar, D. J. J. Kim Y. Proteomic analysis of and secretome by a of and Scholar, A. F. proteomics of secreted Mol. Biol. Scholar, A. J. C. 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