Enriching Cysteine-Containing Peptides Using a Sulfhydryl-Reactive Alkylating Reagent with a Phosphonic Acid Group and Immobilized Metal Affinity Chromatography

The
reduction of disulfide bonds and their subsequent alkylation
are 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 the SH-SY5Y human cell line following 24 h treatments with two
proteasome inhibitors (bortezomib and MG-132) in a tandem mass tag
(TMT)pro9-plex experiment. We acquire three datasets(1) Cys-peptide
enriched, (2) the unbound complement, and (3) the non-depleted controland
compare the peptides and proteins quantified in each dataset, with
emphasis on Cys-containing peptides. The data show that enrichment
using 6C-Cys phosphonate adaptable tag (6C-CysPAT) can quantify over
38,000 Cys-containing peptides in 5 h with >90% specificity. In
addition,
our combined dataset provides the research community with a resource
of over 9900 protein abundance profiles exhibiting the effects of
two different proteasome 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.