Structural Proteomics by Crosslinking Mass Spectrometry

Ph.D. research, University of Victoria / Genome BC Proteomics Centre

← Research

Before moving into quantitative and clinical proteomics, I worked in Christoph Borchers’ lab developing and applying cross-linking mass spectrometry (XL-MS) — a technique that captures spatial constraints between residues in a protein or complex, giving structural information in systems too dynamic, too large, or too intractable for crystallography or cryo-EM alone. My work spanned reagent and software development, chaperone mechanism, intrinsically disordered proteins, and applied structural biology of pathogens.

Reagent & software development

I contributed to the development of isotopically-coded, MS-cleavable and photo-reactive crosslinking reagents designed to make crosslinked peptides easier to distinguish from the vast excess of unmodified peptide background, and to DXMSMS Match, software for automated analysis of the resulting LC-MS/MS data. A related method I developed combined non-specific proteinase K digestion with higher-order (three-peptide) crosslink analysis to substantially improve disulfide bond identification in proteins.

Chaperone mechanism & disordered proteins

Using XL-MS together with hydrogen-deuterium exchange, I mapped the client-binding site of the ATP-independent chaperone Spy, contributing to a broader study of how chaperone flexibility governs client binding (“Super Spy” variants, eLife), and contributed structural mass spectrometry data toward the characterization of client binding by a 2-cysteine peroxiredoxin chaperone (Nature Communications). A parallel line of work used crosslinking-guided discrete molecular dynamics simulations to characterize the native structure and oligomerization of the intrinsically disordered protein tau (Structure).

Applied structural biology

I applied these methods to pathogen structural biology — the export mechanism of EspB from the M. tuberculosis ESX-1 type VII secretion system (Structure), and the interdomain organization of the Plasmodium falciparum surface protein Pf12 (Journal of Biological Chemistry) — as well as to the in-organello mitochondrial protein interactome and the structural characterization of prion protein aggregates by isotopically-coded oxidative labeling.

Into bioinformatics

Between 2020 and 2022 I moved into a bioinformatics-focused role in the same department, building R-based tools for proteogenomics: {hgvsr}, an R package for interpreting HGVS variant nomenclature and evaluating the resulting peptides for LC-MS suitability, and PIGQpipe, a {shiny} web application for predicting variant-peptide coverage across different proteases. That work is the direct bridge to the proteogenomic and isoform-resolved DIA-MS methods I now apply at Columbia.

Crosslinking data and mass spectrometry expertise from this period were also acknowledged in a few externally led structural studies from Patrick Cramer’s lab, including the RNA Polymerase II–Mediator core initiation complex (Plaschka et al., Nature) and the Mediator middle module (Larivière et al., Nucleic Acids Research).

Selected publications

Asterisked authors contributed equally.

  • Makepeace KAT*, Mohammed Y*, Rudashevskaya EL*, et al. Improving identification of in-organello protein–protein interactions using an affinity-enrichable, isotopically-coded, and mass spectrometry-cleavable chemical crosslinker. Molecular & Cellular Proteomics, 2020.
  • Makepeace KAT*, Brodie NI*, Popov KI*, et al. Ligand-induced disorder-to-order transitions characterized by structural proteomics and molecular dynamics simulations. Journal of Proteomics, 2020.
  • Popov KI*, Makepeace KAT*, Petrotchenko EV, Dokholyan NV, Borchers CH. Insight into the structure of the “unstructured” tau protein. Structure, 2019.
  • Teixeira F, Tse E, Castro H, Makepeace KAT, et al. Chaperone activation and client binding of a 2-cysteine peroxiredoxin. Nature Communications, 2019.
  • Groitl B, Horowitz S, Makepeace KAT, et al. Protein unfolding as a switch from self-recognition to high-affinity client binding. Nature Communications, 2016.
  • Makepeace KAT, Serpa JJ, Petrotchenko EV, Borchers CH. Comprehensive identification of disulfide bonds using non-specific proteinase K digestion and CID-cleavable crosslinking analysis methodology. Methods, 2015.
  • Solomonson M, Setiaputra D, Makepeace KAT, et al. Structure of EspB from the ESX-1 type VII secretion system and insights into its export mechanism. Structure, 2015.
  • Petrotchenko EV, Makepeace KAT, Borchers CH. DXMSMS Match program for automated analysis of LC-MS/MS data obtained using isotopically-coded CID-cleavable crosslinking reagents. Current Protocols in Bioinformatics, 2014.
  • Brodie NI, Makepeace KAT, Petrotchenko EV, Borchers CH. Isotopically-coded short-range hetero-bifunctional photo-reactive crosslinkers for studying protein structure. Journal of Proteomics, 2014.
  • Quan S, Wang L, Petrotchenko EV, Makepeace KAT, et al. Super Spy variants implicate flexibility in chaperone action. eLife, 2014.
  • Serpa JJ, Makepeace KAT, Borchers TH, Wishart DS, Petrotchenko EV, Borchers CH. Using isotopically-coded hydrogen peroxide as a surface modification reagent for the structural characterization of prion protein aggregates. Journal of Proteomics, 2013.
  • Tonkin ML, Arredondo SA, Loveless BC, Serpa JJ, Makepeace KAT, et al. Structural and biochemical characterization of Plasmodium falciparum 12 (Pf12) reveals a unique interdomain organization. Journal of Biological Chemistry, 2013.

For the complete list, see my Google Scholar profile or ORCID record.