Quantification of proteins by data independent acquisition: Performance assessment of the Hi3 methodology.

Proteomics greatly benefited from the development of mass spectrometry. Over the last years, data-independent acquisitions increased in popularity in an effort to provide routine label free quantitative information. In this report, the performance of the Hi3 label free method was assessed based on the analysis of a plasma-derived protein mixture. The following parameters of the method (CVs) were determined: repeatability 13.8%, intermediate precision 27.6%, bias 32.3% and linearity observed over 3 orders of magnitude. Finally an accuracy of 42.5% corresponding to a confidence interval within 2 fold the expected protein abundance should be a good approximation of the method performance.

Mass spectrometry based analysis of human plasma-derived factor X revealed novel post-translational modifications.

Human coagulation factor X is a central component of the blood coagulation cascade that converts, under its activated form, prothrombin into thrombin. Generation of thrombin is the final step of the clotting cascade that leads to the clot by polymerization of fibrinogen molecules into a fibrin network. Today, research of new by-passing agents of the coagulation may contribute to an increased interest for human factor X, which may, in consequence, lead to the need of a more exhaustive picture of its structural features. Several post-translational modifications of human factor X such as γ-carboxylation/ß-hydroxylation of the N-terminal light chain and N-/O-glycosylation of the activation peptide have been described. But, so far as we know, no comprehensive studies of its post-translational modifications have been reported. In this article we report an exhaustive structural analysis of human factor X by mass spectrometry using successive protein and peptide mapping. Surprisingly, human factor X was found to be mostly O-glucosylated on its light chain at Ser106 position, Ser9 of its activation peptide is phosphorylated at about 30% and its C-terminal heavy chain is fully O-glycosylated at Thr249 by a mucin-type O-glycan (HexNAc-Hex-NeuAc). The knowledge of these post-translational modifications is mandatory for the development of recombinant molecules.

Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry.

We describe a fast and informative method to investigate the posttranslational modifications of monoclonal antibodies (MAbs). The MAb is first digested by a specific enzyme that cleaves heavy chains under the hinge domain. After reduction of disulfide bridges, three polypeptide chains of approximately 25 kDa are released and analyzed by liquid chromatography-mass spectrometry (LC-MS). By bisecting the heavy chains prior to MS analysis, this method provides a better MS resolution and facilitates the study of the N-linked glycans as well as of other modifications (loss of C-terminal lysine, pyroglutamination, and oxidation). The sample preparation and analysis can be performed within few hours.