Bait Correlation Improves Interactor Identification by Tandem Mass Tag-Affinity Purification-Mass Spectrometry

The quantitative multiplexing capacity
of isobaric tandem mass
tags (TMT) has increased the throughput of affinity purification mass
spectrometry (AP-MS) to characterize protein interaction networks
of immunoprecipitated bait proteins. However, variable bait levels
between replicates can convolute interactor identification. We compared
the Student’s t-test and Pearson’s R correlation as methods to generate t-statistics
and assessed the significance of interactors following TMT-AP-MS.
Using a simple linear model of protein recovery in immunoprecipitates
to simulate reporter ion ratio distributions, we found that correlation-derived t-statistics protect against bait variance while robustly
controlling type I errors (false positives). We experimentally determined
the performance of these two approaches for determining t-statistics under two experimental conditions: irreversible prey
association to the Hsp40 mutant DNAJB8H31Q followed by
stringent washing, and reversible association to 14-3-3ζ with
gentle washing. Correlation-derived t-statistics
performed at least as well as Student’s t-statistics
for each sample and with substantial improvement in performance for
experiments with high bait-level variance. Deliberately varying bait
levels over a large range fails to improve selectivity but does increase
the robustness between runs. The use of correlation-derived t-statistics should improve identification of interactors
using TMT-AP-MS. Data are available via ProteomeXchange with identifier
PXD016613.