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 impractical, as each biological sample requires weeks of measurement time.
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.
Native protein extracts from biological triplicates of HeLa cells (CCL2 or Kyoto variants) were separated by SEC into 70 fractions and analysed by SWATH-MS. We used a 21-minute gradient on the EvoSep One HPLC system that uses embedded gradients to minimize overhead. Data were analysed using OpenSWATH and Spectronaut and protein complex reorganization was determined using CCprofiler and SECAT.
Over 3 replicates each from 2 conditions we detected 4,065 proteins with SEC elution features of which 2,026 had multiple features indicating that the protein is present in multiple distinct assembly states. Querying the data for CORUM complexes, we saw evidence for 590 complexes (5% FDR). Comparison with a prior study using 2 hour gradients showed we retain the majority of information at the protein (99%) and protein complex (95%) levels with >10-fold increase in speed. Quantitative analysis showed that while many proteins are differentially abundant between HeLa variants, changes in protein complexes are less prevalent consistent with complex level-buffering effects. Preliminary data using the newly developed diaPASEF method for this analysis will also be presented.
We have optimized SEC-SWATH-MS to analyse 1 biological sample per day. The dramatic increase in throughput enables the characterization of proteome complex organization with minimal information loss, extended scope, and broad applicability.