Tadi Surendar, Sharp Joshua S
Department of Biomolecular Sciences, University of Mississippi, Oxford, Mississippi, 38677, USA.
J Biomol Tech. 2019 Dec;30(4):50-57. doi: 10.7171/jbt.19-3004-002.
Methionine oxidation plays a critical role in many processes of biologic and biomedical importance, including cellular redox responses and stability of protein pharmaceuticals. Bottom-up methods for analysis of methionine oxidation can suffer from incomplete sequence coverage, as well as an inability to readily detect correlated oxidation between 2 or more methionines. However, the methodology for quantifying protein oxidation in top-down analyses is lacking. Previous work has shown that electron transfer dissociation (ETD)-based tandem mass spectrometry (MS/MS) fragmentation offers accurate and precise quantification of amino acid oxidation in peptides, even in complex samples. However, the ability of ETD-based MS/MS fragmentation to accurately quantify amino acid oxidation of proteins in a top-down manner has not been reported. Using apomyoglobin and calmodulin as model proteins, we partially converted methionines into methionine sulfoxide by incubation in HO. Using top-down ETD-based fragmentation, we quantified the amount of oxidation of various ETD product ions and compared the quantified values with those from traditional bottom-up analysis. We find that overall quantification of methionine oxidation by top-down MS/MS ranges from good agreement with traditional bottom-up methods to vast differences between the 2 techniques, including missing oxidized product ions and large differences in measured oxidation quantities. Care must be taken in transitioning ETD-based quantitation of oxidation from the peptide level to the intact protein level.
甲硫氨酸氧化在许多具有生物学和生物医学重要性的过程中起着关键作用，包括细胞氧化还原反应和蛋白质药物的稳定性。用于分析甲硫氨酸氧化的自下而上方法可能存在序列覆盖不完整的问题，以及无法轻易检测两个或更多甲硫氨酸之间的相关氧化。然而，自上而下分析中用于量化蛋白质氧化的方法尚不存在。先前的工作表明，基于电子转移解离（ETD）的串联质谱（MS/MS）碎裂能够对肽段中的氨基酸氧化进行准确和精确的量化，即使在复杂样品中也是如此。然而，基于ETD的MS/MS碎裂以自上而下方式准确量化蛋白质氨基酸氧化的能力尚未见报道。我们以脱辅基肌红蛋白和钙调蛋白作为模型蛋白，通过在过氧化氢中孵育将部分甲硫氨酸转化为甲硫氨酸亚砜。使用基于自上而下ETD的碎裂方法，我们量化了各种ETD产物离子的氧化量，并将量化值与传统自下而上分析得到的值进行比较。我们发现，通过自上而下的MS/MS对甲硫氨酸氧化进行的总体量化，与传统自下而上方法的一致性范围从良好到两种技术之间存在巨大差异，包括缺失氧化产物离子以及测量的氧化量存在很大差异。在将基于ETD的氧化定量从肽段水平转换到完整蛋白质水平时必须谨慎。
