Oral Presentation 25th Annual Lorne Proteomics Symposium 2020

Complex-centric proteome profiling in one day with SEC-SWATH-MS (#81)

Ben Collins 1
  1. Queen's University Belfast, Belfast, ANTRIM, United Kingdom

Cellular functions are rarely attributable to a single molecule but rather to sets of molecules organized into modules such as protein complexes. Methods based on protein correlation profiling (PCP), such as size exclusion chromatography-SWATH mass spectrometry (SEC-SWATH-MS), provide rich information on the state of cellular protein complexes but remain somewhat impractical, as each biological sample requires ~weeks of measurement time.
By employing short (21 minute) gradient analysis we aimed to establish an SEC-SWATH-MS strategy operating at a rate of ~1 biological sample analysed per day while minimizing loss of information. Comparison with a prior study using 2 hour gradients showed we retain the majority of information at the protein and protein complex levels with >10-fold increase in speed. We have applied this method in two perturbed systems, (i) a comparison between the ccl2 and Kyoto variants of the HeLa cell line, and (ii) a comparison between THP-1 cells in monocytic, differentiated macrophage, or LPS stimulated states, revealing a variety of re-organized protein complexes.
The dramatic increase in throughput enables the unbiased characterization of proteome complex organization with minimal information loss, extended scope, and broad applicability.