Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Size exclusion chromatography protein correlation profiling paired with crosslinking mass spectrometry for enhanced identification of yeast protein-protein interactions (#848)

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

Whole proteome crosslinking mass spectrometry is now becoming feasible. The latest mass spectrometry approaches have dramatically improved crosslink identification in complex samples. However, for optimal large-scale analysis, major considerations remain how to decomplexify whole cell lysates to maximise crosslink identification and how to bring biological context to all the interactions that are found. Here we address both considerations by combining size exclusion chromatography and protein correlation profiling with crosslinking mass spectrometry. The application of size exclusion chromatography to intact native yeast complexes firstly decomplexifies the sample to enhance data acquisition and identifications within the mass spectrometer and, secondly, maintains biological context for protein-protein interactions. Wild-type yeast was subjected to offline size exclusion high-performance liquid chromatography, followed by crosslinking with the mass spectrometry cleavable crosslinker DSSO. 70 fractions were then analysed using LC-MS/MS CID+ETD/MS CID on a Fusion Lumos, for 180 min each fraction. Crosslink identification was performed using Proteome Discoverer 2.2 node, XlinkX. Across 70 fractions we detected 2,217 crosslinks representing 1,944 protein-protein interactions (FDR 5%). The yeast nucleosome, RNA polymerase, ribosome, oligosaccharyltranferase complex and vacuolar ATPase are examples of complexes detected in the experiment. Preliminary benchmarking analyses have shown that 208 of the total crosslinks found can be mapped onto known crystallographic structures. Of these, 90.4% are within the distance constraints of the crosslinker. Interestingly, mapped crosslinks exhibited a wide range of XlinkX scores (30-400). Therefore, score may not present an absolute metric for crosslink quality. Overall, our approach represents a novel approach for detecting crosslinks from intact complexes and has generated a foundational dataset of crosslinked protein-protein interactions in yeast.