Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation

Amber E. Boyatzis, Scott D. Bringans, Matthew J. Piggott, Marisa N. Duong, Richard J. Lipscombe, Peter G. Arthur

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Oxidative stress, caused by reactive oxygen and nitrogen species (BONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (similar to 44%) of gels and proteolytic digestion buffers (similar to 20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.

Original languageEnglish
Pages (from-to)2004-2015
Number of pages12
JournalJournal of Proteome Research
Volume16
Issue number5
DOIs
Publication statusPublished - May 2017

Cite this

Boyatzis, Amber E. ; Bringans, Scott D. ; Piggott, Matthew J. ; Duong, Marisa N. ; Lipscombe, Richard J. ; Arthur, Peter G. / Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation. In: Journal of Proteome Research. 2017 ; Vol. 16, No. 5. pp. 2004-2015.
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Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation. / Boyatzis, Amber E.; Bringans, Scott D.; Piggott, Matthew J.; Duong, Marisa N.; Lipscombe, Richard J.; Arthur, Peter G.

In: Journal of Proteome Research, Vol. 16, No. 5, 05.2017, p. 2004-2015.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation

AU - Boyatzis, Amber E.

AU - Bringans, Scott D.

AU - Piggott, Matthew J.

AU - Duong, Marisa N.

AU - Lipscombe, Richard J.

AU - Arthur, Peter G.

PY - 2017/5

Y1 - 2017/5

N2 - Oxidative stress, caused by reactive oxygen and nitrogen species (BONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (similar to 44%) of gels and proteolytic digestion buffers (similar to 20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.

AB - Oxidative stress, caused by reactive oxygen and nitrogen species (BONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (similar to 44%) of gels and proteolytic digestion buffers (similar to 20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.

KW - mass spectrometry

KW - gel electrophoresis

KW - oxidative stress

KW - proteomics

KW - sample preparation

KW - MASS-SPECTROMETRY

KW - QUANTITATIVE PROTEOMICS

KW - DIFFERENTIAL ALKYLATION

KW - CYSTEINE OXIDATION

KW - N-ETHYLMALEIMIDE

KW - S-NITROSYLATION

KW - LABEL-FREE

KW - IN-VIVO

KW - IDENTIFICATION

KW - SUCCINIMIDE

U2 - 10.1021/acs.jproteome.6b01060

DO - 10.1021/acs.jproteome.6b01060

M3 - Article

VL - 16

SP - 2004

EP - 2015

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 5

ER -