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Article Dans Une Revue Molecular and Cellular Proteomics Année : 2010

Analysis of Human C1q by Combined Bottom-up and Top-down Mass Spectrometry

Résumé

C1q is a subunit of the C1 complex, a key player in innate immunity that triggers activation of the classical complement pathway. Featuring a unique structural organization and comprising a collagen-like domain with a high level of post-translational modifications, C1q represents a challenging protein assembly for structural biology. We report for the first time a comprehensive proteomics study of C1q combining bottom-up and top-down analyses. C1q was submitted to proteolytic digestion by a combination of collagenase and trypsin for bottom-up analyses. In addition to classical LC-MS/MS analyses, which provided reliable identification of hydroxylated proline and lysine residues, sugar loss-triggered MS3 scans were acquired on an LTQ-Orbitrap (Linear Quadrupole Ion Trap-Orbitrap) instrument to strengthen the localization of glucosylgalactosyl disaccharide moieties on hydroxylysine residues. Top-down analyses performed on the same instrument allowed high accuracy and high resolution mass measurements of the intact full-length C1q polypeptide chains and the iterative fragmentation of the proteins in the MSn mode. This study illustrates the usefulness of combining the two complementary analytical approaches to obtain a detailed characterization of the post-translational modification pattern of the collagen-like domain of C1q and highlights the structural heterogeneity of individual molecules. Most importantly, three lysine residues of the collagen-like domain, namely Lys59 (A chain), Lys61 (B chain), and Lys58 (C chain), were unambiguously shown to be completely unmodified. These lysine residues are located about halfway along the collagen-like fibers. They are thus fully available and in an appropriate position to interact with the C1r and C1s protease partners of C1q and are therefore likely to play an essential role in C1 assembly.

Dates et versions

hal-01967565 , version 1 (31-12-2018)

Identifiants

Citer

Delphine Pflieger, Cédric Przybylski, Florence Gonnet, Jean-Pierre Le Caer, Thomas Lunardi, et al.. Analysis of Human C1q by Combined Bottom-up and Top-down Mass Spectrometry. Molecular and Cellular Proteomics, 2010, 9 (4), pp.593-610. ⟨10.1074/mcp.M900350-MCP200⟩. ⟨hal-01967565⟩
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