Beyond Abundance: Quantifying and Interpreting Post-translational Modification Stoichiometry
Year: 2026
Type: conference-paper
Abstract: Mass spectrometry quantifies post-translational modifications (PTMs) across tens of thousands of sites in a single experiment, yet measurements are typically reported as the relative abundance of a modified peptide. This abundance reflects both the amount of the parent protein and the fractional stoichiometry of the modification, which a relative measurement alone cannot distinguish. A twofold increase in a phosphopeptide could reflect a change in fractional stoichiometry from 0.1% to 0.2% or from 30% to 60%, with potentially very different functional consequences. Fractional stoichiometry, the fraction of protein molecules carrying a modification at a given site, is measurable for several PTM classes but remains rarely reported. Using a streamlined differential-labelling workflow, we quantify free-thiol occupancy of primary human cells at more than 10,000 cysteines across ~4,600 proteins in a single experiment, measuring the absolute reduced fraction of each residue. Cysteine reduced-state stoichiometry spans the full range, with a median of ~60% reduced, and varies as much within proteins as between them: among proteins with multiple measured cysteines, the median within-protein range exceeds 20 percentage points, and several hundred proteins span more than 40. Modification state is, therefore, a property of residues, not proteins. This presents a fundamental challenge for current analytical frameworks. Network medicine, for example, maps molecular measurements onto the protein interactome, but a network with one node per protein cannot represent residue-level state: every site on a protein inherits the same neighbours, regardless of its modification state. We propose that absolute, residue-resolved stoichiometry requires a correspondingly residue-resolved framework—one that incorporates structural proximity, interaction interfaces, and coordinated changes in stoichiometry—to move from cataloguing PTMs to understanding how modification state is organized across the proteome.
Authors: Niv Vigder, João Paulo, Joseph Loscalzo
Institutions: Brigham and Women's Hospital, Harvard University
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: gold
OpenAlex ID: W7203894101
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