Enriching cysteine-containing peptides using a sulfhydryl-reactive alkylating reagent with a phosphonic acid group, and immobilized metal affinity chromatography

ABSTRACT: The reduction of disulfide bonds and their subsequent alkylation is commonplace in typical proteomics workflows. Here, we highlight a sulfhydryl-reactive alkylating reagent with a phosphonic acid group (iodoacetamido-LC-phosphonic acid, 6C-CysPAT) that facilitates the enrichment of cysteine-containing peptides for isobaric tag-based proteome abundance profiling. Specifically, we profile the proteome of SH-SY5Y cells following 24 hr treatments with two proteasome inhibitors (bortezomib and MG-132) in a TMTpro9-plex experiment. We acquired three datasets - 1) Cys-enriched, 2) Cys-depleted, and 3) non-depleted - and compared the peptides and proteins quantified in each dataset, with emphasis on Cys-containing peptides. The data show that enrichment using 6C-CysPAT quantified over 38,000 Cys-containing peptides in 5 hr with >90% specificity. In addition, our combined dataset provides the research community with a resource of over 9,900 protein abundance profiles that exhibit the effects of two different proteosome inhibitors. Overall, the seamless incorporation of alkylation by 6C-CysPAT into a current TMT-based workflow permits the enrichment of a Cys-containing peptide subproteome. The acquisition of this “Mini-Cys” dataset can be used to preview and assess the quality of a deep, fractionated dataset.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Joao Paulo  

LAB HEAD: Joao A. Paulo

PROVIDER: PXD038382 | Pride | 2023-12-28

REPOSITORIES: Pride