Dataset Information

Mining the secretome of C2C12 muscle cells

ABSTRACT: Secretome analysis faces several challenges, including detection of low abundant proteins and the discrimination of bona fide secreted proteins from false-positive identifications stemming from cell leakage or serum. Here, we developed a two-step secretomics approach and applied it for the analysis of secreted proteins of C2C12 skeletal muscle cells as the skeletal muscle has been identified as an important endocrine organ secreting myokines as active signaling molecules. First, we compared culture supernatants and corresponding cell lysates by high-resolution MS-based proteomics and label-free quantification. We found 672 protein groups as candidate secreted proteins, as they showed a higher abundance in the secretome. By Brefeldin A mediated blocking of classical secretory processes, we estimate a sensitivity of > 80% for the detection of classical secreted proteins by our experimental approach. In the second step, peptide level information was integrated with UniProt based protein information by the bioinformatics tool “lysate and secretome peptide feature plotter” (LSPFP) to detect proteolytic protein processing events which might occur during secretion e.g. As a proof on concept, we identified truncations of the cytoplasmic part of the protein Plexin-B2. Our workflow provides an efficient combination of experimental workflow and data analysis to identify putative secreted and proteolytic processed proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Permanent Cell Line Cell, Cell Culture, Muscle Cell

SUBMITTER: Gereon Poschmann

LAB HEAD: Gereon Poschmann

PROVIDER: PXD007527 | Pride | 2018-01-24

REPOSITORIES: Pride

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Publications

Mining the Secretome of C2C12 Muscle Cells: Data Dependent Experimental Approach To Analyze Protein Secretion Using Label-Free Quantification and Peptide Based Analysis.

Grube Leonie L Dellen Rafael R Kruse Fabian F Schwender Holger H Stühler Kai K Poschmann Gereon G

Journal of proteome research 20180124 2

Secretome analysis faces several challenges including detection of low abundant proteins and the discrimination of bona fide secreted proteins from false-positive identifications stemming from cell leakage or serum. Here, we developed a two-step secretomics approach and applied it to the analysis of secreted proteins of C2C12 skeletal muscle cells since the skeletal muscle has been identified as an important endocrine organ secreting myokines as signaling molecules. First, we compared culture su ...[more]

PMID: 29322779

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Project description:Background Loss of skeletal muscle mass in cancer cachexia is recognized as an independent predictor of mortality. Mechanisms involved in this wasting process and parameters for early diagnosis are not yet clearly defined. As skeletal muscle is considered as a secretory organ, the aim of this present experimental work was to characterize the changes in the putative muscle secretome associated with cancer-induced cachexia to gain a better understanding of cellular mechanisms involved and to identify secreted proteins which might reflect this wasting process. Methods We investigated first the changes in the muscle proteome associated with cancer-induced cachexia by using differential label-free proteomic analysis on muscle of the C26 mouse model. The differentially abundant proteins were then submitted to sequential bioinformatic secretomic analysis in order to identify potentially secreted proteins. Multiple reaction monitoring and Western blotting were used to verify the presence of candidate proteins at the circulating level. Finally, we investigated the regulation of the production of these secreted proteins by muscle in vitro and in vivo. Results Our results revealed a dramatic increased muscular production (2-to 25-fold) of several acute phase reactants (APR: haptoglobin, serpina3n, complement C3, serum amyloid A1) which are released in the circulation during C26 cancer cachexia. This observation was confirmed in two other preclinical models of cancer cachexia as well as in cancer patients. The muscular origin of these APR was demonstrated by their increased expression in skeletal muscle and myotubes. Our results showed also that IL-6 plays a major role in the muscular induction of these APR in vivo, while glucocorticoids and pro-inflammatory cytokines stimulate directly their increased expression in muscle cells in vitro. Conclusions Muscle wasting caused by cancer is associated with marked changes in muscle secretome. Our study demonstrates a marked increased production of APR by skeletal muscle in pre-clinical models of cancer cachexia and in cancer patients. Further studies are required to unravel the potential role of these proteins in muscle atrophy and their interest as biomarkers of cancer cachexia.

Project description:Background Loss of skeletal muscle mass in advanced cancer is recognized as an independent predictor of mortality. Mechanisms involved in this wasting process and parameters for early diagnosis are still lacking. As skeletal muscle is considered as a secretory organ, the aim of this present experimental work was to characterize the changes in muscle proteome and secretome associated with cancer-induced cachexia to better understand cellular mechanisms involved in this wasting process and to identify secreted proteins which might reflect the ongoing muscle atrophy process. Methods We investigated first the changes in the muscle proteome associated with cancer-induced cachexia by using differential label-free proteomic analysis on muscle of the C26 mouse model. The differentially abundant proteins were submitted to sequential bioinformatic secretomic analysis in order to identify potentially secreted proteins. Selected reaction monitoring and Western blotting were used to verify the presence of candidate proteins at the circulating level. Their muscle source was demonstrated by assessing their gene expression in skeletal muscle and in cultured myotubes. Finally, we also investigated their regulation in muscle cells. Alterations in several molecular pathways potentially involved in muscle atrophy were highlighted using Gene ontology enrichment analyses. Results Our results revealed a dramatic increased production (2-to 25-fold) by the muscle of several acute phase reactants (APR: Haptoglobin, Serpina3n, Complement C3, Serum amyloid A1) which are also released in the circulation during C26 cancer cachexia. Their production was confirmed in other preclinical models of cancer cachexia as well as in cancer patients. The muscular origin of these APR was demonstrated by their increased expression in skeletal muscle and myotubes. Glucocorticoids and pro-inflammatory cytokines contribute directly to their increased expression in muscle cells in vitro, while the role of IL-6 in the muscular induction of these APR was demonstrated in vivo. Conclusions Cancer is associated with marked changes in muscle secretome during muscle wasting. Our study demonstrates a marked increased production of APR by skeletal muscle in pre-clinical models of cancer cachexia and in cancer patients. Further studies are required to unravel the potential role of these proteins in muscle atrophy and their interest as biomarkers of cancer cachexia.

Dataset Information

Mining the secretome of C2C12 muscle cells

Publications

Mining the Secretome of C2C12 Muscle Cells: Data Dependent Experimental Approach To Analyze Protein Secretion Using Label-Free Quantification and Peptide Based Analysis.

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