Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents

Abstract

Reduction and alkylation of cysteine residues is part of virtually any proteomics workflow. Despite its frequent use, up to date no systematic investigation of the impact of different conditions on the outcome of proteomics studies has been performed. In this study, we compared common reduction reagents (dithiothreitol, tris-(2-carboxyethyl)-phosphine, and β-mercaptoethanol) and alkylation reagents (iodoacetamide, iodoacetic acid, acrylamide, and chloroacetamide). Using in-gel digests as well as SAX fractionated in-solution digests of cytosolic fractions of HeLa cells, we evaluated 13 different reduction and alkylation conditions resulting in considerably varying identification rates. We observed strong differences in offsite alkylation reactions at 7 amino acids as well as at the peptide N terminus, identifying single and double adducts of all reagents. Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, we identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. We observed differences of more than 9-fold in numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and noniodine-containing alkylation reagents. This was because of formation of carbamidomethylated and carboxymethylated methionine side chains and a resulting prominent neutral loss during ESI ionization or in MS/MS fragmentation, strongly decreasing identification rates of methionine-containing peptides. We achieved best results with acrylamide as alkylation reagent, whereas the highest numbers of peptide spectral matches were obtained when reducing with dithiothreitol and β-mercaptoethanol for the in-solution and the in-gel digested samples, respectively.

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

“…There have been reports regarding the alkylation at the side chains of other amino acids besides the desired alkylation of cysteine, including aspartic acid, glutamic acid, lysine, histidine, tyrosine, serine, and threonine. 51–53, 64 Even di- and tri-alkylation on the side chains of some amino acid residues of peptides may further occur, 65 but it is less common. In this study, we mainly focused on mono-alkylation at cysteine, histidine, lysine, aspartic acid, glutamic acid, tyrosine, and the peptide N- and C-termini.…”

“…51, 52 Recently, Muller and Winter have reported the systematic evaluation of protein reduction and alkylation, which has also included side reactions on the side chains of tyrosine, serine, and threonine. 53 Therefore, it is critical to push the desired reaction toward completion and minimize the side reactions. Compared to that paper, 53 the current work has several differences: (1) The experimental designs are different.…”

“…53 Therefore, it is critical to push the desired reaction toward completion and minimize the side reactions. Compared to that paper, 53 the current work has several differences: (1) The experimental designs are different. Here we compared only one parameter with all other parameters fixed for each individual experiment, while in that paper, different combinations of reducing and alkylating reagents were compared.…”

Updates of the In‐Gel Digestion Method for Protein Analysis by Mass Spectrometry

“…Our work collates recent advances in tryptic digestion to create beneficial updates of the in‐gel digestion protocol . We show superior protein identification and sequence coverage with reduced side reactions and handling time, when compared with the basic approach.…”

“…Tryptic digestion methods have been extensively improved over the years . Recently, an investigation on reducing and alkylating reagents increased the number of proteins identified and protein sequence coverage by replacing the widely used DTT and iodoacetamide (IAA) reagents …”