Havelund Jesper F, Wojdyla Katarzyna, Davies Michael J, Jensen Ole N, Møller Ian Max, Rogowska-Wrzesinska Adelina
Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark; Department of Molecular Biology and Genetics, Science and Technology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark.
Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark; The Babraham Institute, CB22 3AT Cambridge, United Kingdom.
J Proteomics. 2017 Mar 6;156:40-51. doi: 10.1016/j.jprot.2016.12.019. Epub 2017 Jan 4.
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°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 (+14Da), an oxidised form of methionine - aspartate semialdehyde (-32Da) - and decarboxylated glutamic acid and aspartic acid (-30Da).
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.
蛋白质羰基化是一种与氧化应激、多种疾病和衰老相关的不可逆蛋白质氧化过程。在此，我们描述了一种以肽为中心的方法，用于鉴定和表征蛋白质中多达14种不同类型的羰基化氨基酸。修饰后的残基用生物素酰肼进行衍生化，通过串联质谱进行富集和表征。该方法的优势在于使用热水（95°C）从单体抗生物素蛋白树脂中改进了生物素化肽的洗脱，并通过减少样品制备和色谱过程中的分析物损失提高了灵敏度。首次利用诊断性生物素碎片离子进行MS/MS数据分析，以确定生物素标记位点并提高羰基化肽归属的可信度。在广泛的体外金属离子催化氧化后，我们在牛血清白蛋白中总共鉴定出125个羰基化残基。此外，我们在天然人血浆蛋白样品中的36种蛋白质中确定了133个羰基化位点。优化后的工作流程能够检测到蛋白质中10种迄今未检测到的羰基化氨基酸类型：亮氨酸、缬氨酸、丙氨酸、异亮氨酸、谷氨酰胺、赖氨酸和谷氨酸的醛和酮修饰（+14Da）、甲硫氨酸的氧化形式——天冬氨酸半醛（-32Da）以及脱羧谷氨酸和天冬氨酸（-30Da）。
蛋白质组学工具为在蛋白质水平解码疾病机制提供了一种有前景的方法，并有助于理解羰基化如何影响蛋白质结构和功能。未来研究的挑战是确定氧化残基的确切类型和性质，以更深入地了解细胞和生物体中羰基化的调控机制，并评估它们在疾病中的作用。
