Quantitative assessment of the effects of trypsin digestion methods on immunoprecipitation-mass spectrometry-based protein-protein interaction analysis - part2

ABSTRACT: Immunoprecipitation-mass spectrometry (IP-MS) has become the method of choice for discovering protein-protein interaction (PPI) under native conditions. The success of the IP-MS depends on the efficiency of trypsin digestion and recovery of the tryptic peptides for MS analysis. Several different protocols have been used for trypsin digestion of protein complexes in IP-MS studies, but no systematic studies have been conducted on the impact of trypsin digestion conditions on the identification of PPI. Here, we identified the interactome of transcription factor p65 (RelA) to test five distinct trypsin digestion methods (two using “on-bead”, two using “in-solution”, and one using “in-gel” protocols) and determined which method yielded the best interactome coverage. Our study indicates that high-abundance RelA interactors can be universally identified by all five methods, but the identification of low-abundance RelA interactors is significantly affected by the choice of trypsin digestion methods. We found that eluting RelA complex with sodium dodecyl sulfate (SDS) and followed by filter-aided sample preparation (FASP)/in-solution digestion is the best among the methods that were tested. We also found that different digestion protocols influences the selected reaction monitoring (SRM)-MS quantification of PPIs, suggesting that optimization of trypsin digestion condition may be required for robust study of targeted analysis of PPIs.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yingxin Zhao  

LAB HEAD: Yingxin Zhao, Ph.D.

PROVIDER: PXD006739 | Pride | 2017-07-26

REPOSITORIES: pride