Quantitative Assessment of the Effects of Trypsin Digestion Methods on Affinity Purification–Mass Spectrometry-based Protein–Protein Interaction Analysis

Affinity
purification-mass spectrometry (AP–MS) has become
the method of choice for discovering protein–protein interactions
(PPIs) under native conditions. The success of AP-MS depends on the
efficiency of trypsin digestion and the recovery of the tryptic peptides
for MS analysis. Several different protocols have been used for trypsin
digestion of protein complexes in AP-MS studies, but no systematic
studies have been conducted on the impact of trypsin digestion conditions
on the identification of PPIs. Here, we used NFκB/RelA and Bromodomain-containing
protein 4 (BRD4) as baits and test five distinct trypsin digestion
methods (two using “on-beads,” three using “elution-digestion”
protocols). Although the performance of the trypsin digestion protocols
change slightly depending on the different baits, antibodies and cell
lines used, we found that elution-digestion methods consistently outperformed
on-beads digestion methods. The high-abundance interactors can be
identified universally by all five methods, but the identification
of low-abundance RelA interactors is significantly affected by the
choice of trypsin digestion method. We also found that different digestion
protocols influence the selected reaction monitoring (SRM)–MS
quantification of PPIs, suggesting that optimization of trypsin digestion
conditions may be required for robust targeted analysis of PPIs.