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 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.

Keywords: 6C-CysPAT; FAIMS; Fe3+-NTA; Orbitrap Eclipse; TMTpro; isobaric tagging.

Figure 1.

Experimental overview. Cells were treated with compounds for 24 h and then harvested. Proteins were extracted with ureacontaining buffer, reduced with TCEP, alkylated with 6C-CysPAT, chloroform–methanol-precipitated, and then digested with LysC followed by trypsin. The digested peptides were labeled with TMT and then pooled. The pooled sample was divided in half. One-half was subjected to enrichment using Fe³⁺-NTA, while the other half was not. The non-depleted (“NoDep”) sample and the unbound peptides (“unbound”) from the Fe³⁺-NTA enrichment were fractionated into 12 fractions. These fractions along with the Fe³⁺-NTA-enriched fraction were subjected to LC-FAIMS-MS/MS. The Fe³⁺-NTA-enriched fraction (“enriched”) was analyzed twice, each time with a different set of CVs. BTZ, bortezomib; and fxn, fraction.

Figure 2.

Quantitative comparison of the three datasets. Bar graphs comparing the number of (A) proteins, (B) total and unique peptides, and (C) Cys-containing peptides that were quantified in the Cys-enriched, unbound complement, and non-depleted (“NoDep”) datasets.

Figure 3.

Inventory of peptides in each of the three datasets. Pie charts comparing the number of cysteine-containing, phosphorylated, and unmodified (neither by alkylation nor phosphorylation) peptides in the (A) enriched, (B) unbound complement, and (C) non-depleted datasets.

Figure 4.

Comparison of the two CV sets used for the Cys-enriched (i.e., “enriched”) dataset. The enriched sample was analyzed twice with two different sets of CVs: set 1 (CV = −40, −60, and −80 V) and set 2 (CV = −35, −50, and −65 V). (A) The bar chart compares the number of total and unique peptides quantified by each CV set. (B) The Venn diagram illustrates the overlap of peptides quantified in the two CV sets. (C) The UpSet plot displays the overlap of peptides quantified when using different CVs (filled, connected circles) and the number of peptides that were distinct to each CV (filled circles, no connection).