Abstract
Modern, quantitative proteome biology relies on bottom-up mass spectrometry-based quantification techniques. Current proteomic methods quantify proteins and compare sample conditions with either isotope-defined metabolic or chemical labels that modify select amino acids in the proteome. Covalent modification of proteins with isotope-defined reagents enables protein footprinting techniques to quantify site-specific conformational information, such as the solvent exposure of amino acids on the surface of proteins. However, the current analysis of chemical protein footprinting experiments like covalent protein painting (CPP) misses quantifying a large proportion of peptides because of ambiguities in the position of the label in case more than one amino acid is modified in the peptide. Here, we developed a mass spectrometry-based approach to deconvolute and quantify the relative mass modifications of two lysine sites in the same peptide. We determined at which lysine site the modification is located on the basis of the fragment ion quantification of the isobaric isotopologues. The quantification approach retained the correlative information on the solvent accessibility between the two lysine sites. The new approach increased the overall quantification efficiency by 15% in a large data set comprising 60 different cancer cell lines. This gain in structural information indicated that one conformational state of the protein nucleolin was present in 10 out of 60 cancer cell lines. In summary, deconvoluting the chemical protein footprinting information on peptides with two modified amino acids afforded higher proteome coverage and finer-grained insights into protein structural information.
