posted on 2021-04-29, 08:45authored bySarah
A. Peck Justice, Neil A. McCracken, José F. Victorino, Guihong D. Qi, Aruna B. Wijeratne, Amber L. Mosley
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 the 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
40× 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 an affinity-purified protein complex as an isobaric trigger channel
within a tandem mass tag (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.