Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes
Year: 2021
Type: article
Abstract: ABSTRACT 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 (T m ) calculations for other unrelated proteins in the samples, with a high positive correlation between T m 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.
Source: Analytical Chemistry
Institution: Indiana University School of Medicine
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $5,000
Funders: Thermo Scientific, Indiana University, Indiana Clinical and Translational Sciences Institute, Ralph W. and Grace M. Showalter Research Trust Fund, National Institutes of Health, School of Medicine, Indiana University, National Heart, Lung, and Blood Institute, National Institute of General Medical Sciences, National Institute of Neurological Disorders and Stroke, National Center for Advancing Translational Sciences
Awards: Regulation of RNA Polymerase II transcription by the phosphatase Rtr1, G3754633476, G4437163022, Basic Sciences Studies on Gene Therapy of Blood Diseases, Indiana Clinical and Translational Sciences Institute, G9000048861, Thermal proteome profiling for analysis of protein sequence variants in human genetic disease
OpenAlex ID: W3158930363