Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics

Abstract

Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, the reaction incompletion and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with the commonly used reagents, i.e., dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, or tris(3-hydroxypropyl)phosphine, and alkylation with iodoacetamide, acrylamide, N-ethylmaleimide, or 4-vinylpyridine. By using digested peptides from a yeast whole-cell lysate, the number of proteins and peptides identified were very similar using four different reducing reagents. The results from four alkylating reagents, however, were dramatically different with iodoacetamide giving the highest number of peptides with alkylated cysteine and the lowest number of peptides with incomplete cysteine alkylation and side reactions. Alkylation conditions with iodoacetamide were further optimized. To identify more peptides with cysteine, Thiopropyl-Sepharose 6B resins were used to enrich them, and the optimal conditions were employed for the reduction and alkylation. The enrichment resulted in over three times more cysteine-containing peptides than without enrichment. Systematic evaluation of the reduction and alkylation with different reagents can aid in a better design of bottom-up proteomic experiments.

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

“…Alkylating reagents chemically modify oxidized cysteine residues to prevent disulfide bridges from forming again. The breakdown of the tertiary structure of a protein often helps proteases access substrates that would have been more difficult to access and therefore increases digestion efficiency (Suttapitugsakul, Xiao, Smeekens, & Wu, 2017).…”

Are Granulins Copper Sequestering Proteins?

“…Therefore, to determine how many cystines are involved in coordination, alkylation of free thiols in the Cu(II)bound holoproteins was conducted with iodoacetamide (IAA) for both GRNs. IAA reacts with free thiols to forms acetamide adduct (58 Da) with sulfur 46 . The Cu(II)-bound GRN-3 at pH 7.0 displayed a gaussian distribution centered around ~two alkylated species (6490.8 Da) suggesting that most of the cystines were coordinated to Cu(II) at pH 7.0 (Fig 3a).…”

Modified cysteine S-phosphopeptide standards for mass spectrometry-based proteomics

“…It is important to mention that proteomic samples may contain traces of thiol groups that emerged from various precursors. In general, the presence of free thiols is mainly associated with incomplete alkylation of thiol groups from reducible sulfhydryl modifications (Dong et al 2014;Muller and Winter 2017;Suttapitugsakul et al 2017). Moreover, even traces of S-acylated peptides can generate Cys-peptides as a result of acyl transfer events (Ji et al 2013(Ji et al , 2016.…”

“…In real-world proteomics, cysteine residues of proteins are usually S-carbamidomethylated (Cys→camCys) (Muller and Winter 2017;Suttapitugsakul et al 2017;Svozil and Baerenfaller 2017) and considered to have been permanently modified. Therefore, phosphocysteine targeting re-analysis of mass spectra should apply a non-routine bioinformatic approach, as MS/MS search settings affect the outcome of intact pCys-peptide identification.…”