Oral Presentation 24th Annual Lorne Proteomics Symposium 2019

Large-scale detection of protein-protein interactions via size-exclusion chromatography, protein correlation profiling and crosslinking mass spectrometry (#61)

Daniela-Lee Smith 1 , Gene Hart-Smith 1 , Marc Wilkins 1
  1. UNSW, Sydney, NSW, Australia

Crosslinking mass spectrometry has the potential to detect thousands of protein-protein interactions in a cell or tissue, thus building interaction networks in a single experiment. However, many technical challenges remain before this can be achieved. One major challenge is how to decomplexify whole cell lysates to maximise crosslink identification, but to do this in a manner which preserves biological relevance. Here we aim to address these considerations through combining size-exclusion chromatography of native protein complexes, protein correlation profiling and crosslinking-mass spectrometry. Wild-type yeast lysate was subjected to offline size-exclusion high-performance liquid chromatography, followed by crosslinking with the mass spectrometry-cleavable DSSO crosslinker. Seventy fractions were then analysed by MS/MS/MS for 180 min per fraction, using LC/MS/MS CID+ETD/MS CID on an Orbitrap Fusion Lumos Tribrid. Crosslinked peptides were identified with XlinkX, using Proteome Discoverer 2.2. Across 70 fractions we detected 2217 crosslinks, representing 1944 protein-protein interactions (FDR 5%). The yeast nucleosome, RNA polymerase, the yeast ribosome, the yeast oligosaccharyltransferase complex and vacuolar ATPase are examples of complexes that were found. The scale of this dataset allow for in-depth benchmarking analyses. Therefore, aspects of crosslink discovery, including score, amino acids involved in links, peptide length and type and fragmentation doublets will be discussed. Overall this experiment represents a significant advance in the detection of crosslinks at a large scale and has strong potential to discover novel protein-protein interactions.