RT Journal Article
SR Electronic
T1 Boosting detection of low abundance proteins in thermal proteome profiling experiments by addition of an isobaric trigger channel to TMT multiplexes
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.12.30.424894
DO 10.1101/2020.12.30.424894
A1 Sarah A. Peck Justice
A1 Neil A. McCracken
A1 José F. Victorino
A1 Aruna B. Wijeratne
A1 Amber L. Mosley
YR 2020
UL http://biorxiv.org/content/early/2020/12/31/2020.12.30.424894.abstract
AB The study of low abundance proteins is a challenge to discovery-based proteomics. Mass-spectrometry (MS) applications, such as thermal proteome profiling (TPP) face specific challenges in detection of the whole proteome as a consequence of the use of nondenaturing extraction buffers. TPP is a powerful method for the study of protein thermal stability, but quantitative accuracy is highly dependent on consistent detection. Therefore, TPP can be limited in its amenability to study low abundance proteins that tend to have stochastic or poor detection by MS. To address this challenge, we incorporated an affinity purified protein complex sample at submolar concentrations as an isobaric trigger channel into a mutant TPP (mTPP) workflow to provide reproducible detection and quantitation of the low abundance subunits of the Cleavage and Polyadenylation Factor (CPF) complex. The inclusion of an isobaric protein complex trigger channel increased detection an average of 40x for previously detected subunits and facilitated detection of CPF subunits that were previously below the limit of detection. Importantly, these gains in CPF detection did not cause large changes in melt temperature (Tm) calculations for other unrelated proteins in the samples, with a high positive correlation between Tm estimates in samples with and without isobaric trigger channel addition. Overall, the incorporation of affinity purified protein complex as an isobaric trigger channel within a TMT multiplex for mTPP experiments is an effective and reproducible way to gather thermal profiling data on proteins that are not readily detected using the original TPP or mTPP protocols.Competing Interest StatementThe authors have declared no competing interest.