Proteomics

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Cysteine alkylating methods in shotgun proteomics and their possible effects on methionine residues

ABSTRACT: Neatly every proteomic protocol has a Cysteine reduction and alkylation step in it preceding the trypsin digestion. It needs to be done in order to remove the s-s bonds and prevent proteins from folding, thus, provide better access for trypsin. Among various reagents used for alkylation, iodoacetamide (IAM) seems to be the most common. Although, it has been shown that IAM can cause some undesired side reactions [Chernobrovkin et al., 2015; Muller et al., 2017]. In this work, we have compared the effect of four alkylating agents: iodoacetamide (IAM), 4-vinylpyridine (4-VP), chloroacetamide (CAM) and S-Methyl methanethiosulfonate (MMTS). In addition, we tried to check whether post-treatment with DTT (p-red) is reasonable in the case of irreversible alkylation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Irina Ilina  

LAB HEAD: Sergei A. Moshkovskii

PROVIDER: PXD013973 | Pride | 2020-10-28

REPOSITORIES: Pride

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60_1.mgf Mgf
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60_1_full.pep.xml Pepxml
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Cysteine alkylation methods in shotgun proteomics and their possible effects on methionine residues.

Kuznetsova Ksenia G KG   Levitsky Lev I LI   Pyatnitskiy Mikhail A MA   Ilina Irina Y IY   Bubis Julia A JA   Solovyeva Elizaveta M EM   Zgoda Victor G VG   Gorshkov Mikhail V MV   Moshkovskii Sergei A SA  

Journal of proteomics 20201020

In order to optimize sample preparation for shotgun proteomics, we compared four cysteine alkylating agents: iodoacetamide, chloroacetamide, 4-vinylpyridine and methyl methanethiosulfonate, and estimated their effects on the results of proteome analysis. Because alkylation may result in methionine modification in vitro, proteomics data were searched for methionine to isothreonine conversions, which may mimic genomic methionine to threonine substitutions found in proteogenomic analyses. We found  ...[more]

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