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

Abstract

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 DNAJB8 H31Q followed by stringent washing, and reversible association to 14-3-3z 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 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.

Factors Affecting Protein Recovery During Hsp40 Affinity Profiling

“…Tryptic digests of the eluate were isobarically labeled with TMT tags, quantified by MuDPIT LC-MS, and relative protein abundances inferred from TMT reporter ion ratios for identified peptides. Interaction significance was determined using our previously reported bait correlation method 21 .…”

“…Co-immunoprecipitated proteins are quantified by mass spectrometry using isobaric TMT labeling. We found that proteins that co-immunoprecipitate with Flag DNAJB8 H31Q are generally destabilized compared to the bulk proteome 21 . We used this Hsp40 affinity assay to identify proteins that are destabilized by brief cellular arsenite exposure, finding a series of known arsenite targets, and validated their destabilization by limited proteolysis.…”

“…Flag DNAJB8 WT .CMV2, and Flag DNAJB8 H31Q .CMV2, and EGFP.pDest30 have been reported 21,23 . Constructs were analytically digested and sequenced (Retrogen) to confirm identity.…”

“…Violin plots were generated in R using the ggplot2 library. For bait vs. mock experiments, Pearson’s R-derived t-statistics were used for determination of p-values 21 . q-values (q BH ) were determined from p-values using Storey’s modification of Benjamini-Hochberg’s methodology 31,32 , and adjusted to maintain monotonicity.…”

Hsp40 Affinity to Identify Proteins Destabilized by Cellular Toxicant Exposure

“…[18][19][20][21] We previously used affinity purification and quantitative proteomics to identify hundreds of cellular protein clients of overexpressed human Hsp40 DNAJB8 H31Q with high reproducibility and statistical confidence. [22] Herein we exploit the ability of DNAJB8 H31Q to recognize misfolded protein clients to develop a platform for identifying proteins that are destabilized in response to exogenous stress (Figure 1). We demonstrate this approach in HEK293T cells treated with trivalent arsenic, a toxic metal that causes widespread damage to nucleic acids and proteins, leading to genomic and metabolic instability.…”