Affinity Purification Trigger with mTPP

ABSTRACT: We have applied a BASIL-like approach to increase the signal of low abundance peptides to the study of protein complexes. To accomplish this, we incorporated an affinity purified cleavage and polyadenylation factor (CPF) complex as a “trigger” into our mTPP workflow at a trigger sample to lowest heat-treated mTPP sample ratio of ~1:8 and ~1:50. We saw a significant increase in the abundance of CPF complex members, including those that were not readily identified without the trigger. Importantly, addition of a trigger into our mTPP workflow does not appear to have a significant impact on the melt temperature (Tm) calculation of proteins detected both with and without the trigger. Use of purified protein complex trigger channels in TPP studies, and potentially other global proteomics applications, will improve the ability to perform proteomic analysis of low abundance protein complexes. As many biologically-relevant, as well as disease-relevant, protein complexes are of relatively low abundance in the cell, improving the detection of such proteins in proteomics experiments would be beneficial to increasing our under-standing of the critical cellular processes these complexes play roles in.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Sarah Peck Justice  

LAB HEAD: Amber L. Mosley

PROVIDER: PXD020689 | Pride | 2021-09-09

REPOSITORIES: Pride