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bioRxiv · 10.1101/2023.01.12.523793

SEC-TMT facilitates quantitative differential analysis of protein interaction networks

Doron-Mandel, E.·Bokor, B. J.·Ma, Y.·Street, L. A.·Tang, L. C.·Abdou, A. A.·Shah, N. H.·Rosenberger, G.·Jovanovic, M.
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Abstract

A proteins molecular interactions and post-translational modifications (PTMs), such as phosphorylation, can be co-dependent and reciprocally co-regulate each other. Although this interplay is central for many biological processes, a systematic method to simultaneously study assembly-states and PTMs from the same sample is critically missing. Here, we introduce SEC-MX (Size Exclusion Chromatography fractions MultipleXed), a global quantitative method combining Size Exclusion Chromatography and PTM-enrichment for simultaneous characterization of PTMs and assembly-states. SEC-MX enhances throughput, allows phosphopeptide enrichment, and facilitates quantitative differential comparisons between biological conditions. Applying SEC-MX to HEK293 and HCT116 cells, we generated a proof-of-concept dataset mapping thousands of phosphopeptides and their assembly-states. Our analysis revealed intricate relationships between phosphorylation events and assembly-states and generated testable hypotheses for follow-up studies. Overall, we establish SEC-MX as a valuable tool for exploring protein functions and regulation beyond abundance changes.

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Doron-Mandel, E., Bokor, B. J., Ma, Y., Street, L. A., Tang, L. C., Abdou, A. A., Shah, N. H., Rosenberger, G., Jovanovic, M.. 2023-01-12. SEC-TMT facilitates quantitative differential analysis of protein interaction networks. https://doi.org/10.1101/2023.01.12.523793

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