Correlative Analysis of Amino Acids Sites for Structure Elucidation with Covalent Protein Painting

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.