期刊
JOURNAL OF PROTEOMICS
卷 156, 期 -, 页码 40-51出版社
ELSEVIER
DOI: 10.1016/j.jprot.2016.12.019
关键词
Protein oxidation; Biotin-hydrazide; Metal ion-catalysed oxidation; Oxidative stress
资金
- Danish Council for Independent Research - Natural Sciences (FNU)
- Graduate School of Science and Technology, Aarhus University
- Sino-Danish Center For Education and Research
- Department of Biochemistry and Molecular Biology
- University of Southern Denmark
- Novo Nordisk Foundation [NNF130C0004294]
- VILLUM Foundation to the VILLUM Center for Bioanalytical Sciences (ONJ)
- Novo Nordisk Fonden [NNF13OC0004294] Funding Source: researchfish
- Villum Fonden [00007292] Funding Source: researchfish
Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95 degrees C) and increased sensitivity achieved by reduction of analyte losses during sample preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid ( + 14 Da), an oxidised form of methionine - aspartate semialdehyde (-32 Da) - and decarboxylated glutamic acid and aspartic acid (-30 Da). Biological significance: Proteomic tools provide a promising way to decode disease mechanisms at the protein level and help to understand how carbonylation affects protein structure and function. The challenge for future research is to identify the type and nature of oxidised residues to gain a deeper understanding of the mechanism(s) governing carbonylation in cells and organisms and assess their role in disease. (C) 2017 Elsevier B.V. All rights reserved.
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