SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site

Guillaume Gabant (1) , Alain Boyer (1) , Martine Cadene (1)

1 CBM - Centre de biophysique moléculaire

Protein modifications, whether chemically induced or post-translational (PTMs), play an essential role for the biological activity of proteins. Understanding biological processes and alterations thereof will rely on the quantification of these modifications on individual residues. Here we present SSPaQ, a subtractive method for the parallel quantification of the extent of modification at each possible site of a protein. The method combines uniform isotopic labeling and proteolysis with MS, followed by a segmentation approach, a powerful tool to refine the quantification of the degree of modification of a peptide to a segment containing a single modifiable amino acid. The strength of this strategy resides in: (1) quantification of all modifiable sites in a protein without prior knowledge of the type(s) of modified residues; (2) insensitivity to changes in the solubility and ionization efficiency of peptides upon modification; and (3) detection of missed cleavages caused by the modification for mitigation. The SSPaQ method was applied to quantify modifications resulting from the interaction of human phosphatidyl ethanolamine binding protein 1 (hPEBP1), a metastasis suppressor gene product, with locostatin, a covalent ligand and antimigratory compound with demonstrated activity towards hPEBP1. Locostatin is shown to react with several residues of the protein. SSPaQ can more generally be applied to induced modification in the context of drugs that covalently bind their target protein. With an alternate front-end protocol, it could also be applied to the quantification of protein PTMs, provided a removal tool is available for that PTM

Domaines: Sciences du Vivant [q-bio]; Biochimie, Biologie Moléculaire; Biologie structurale [q-bio.BM]; Biochimie [q-bio.BM]; Chimie analytique

Guillaume Gabant, Alain Boyer, Martine Cadene. SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site. Journal of The American Society for Mass Spectrometry, 2016, 27 (8), pp.1328 - 1343. ⟨10.1007/s13361-016-1416-y⟩. ⟨hal-01406961⟩