Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas 76107, USA.
J Mass Spectrom. 2011 Oct;46(10):976-85. doi: 10.1002/jms.1978.
Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, α, β-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-induced carbonylation. We report a method that uses light (H(12)CHO) and heavy (D(13)CDO) isotopic variant of formaldehyde to differentially label primary amines (N-termini and ε-amino group of lysines) in peptides through reductive methylation and, combined with selective enrichment of modified peptides, permits comparison of the extent of carbonylation in two samples after mixing for simultaneous liquid chromatography-mass spectrometry. Specifically, dimethyl-labeled peptide carbonyls were fractionated from unmodified peptides using solid-phase hydrazide chemistry to immobilize them to porous glass beads and, after removing the unmodified peptides by thoroughly washing the beads, subsequently recover them by acid-catalyzed hydrolysis. The method was developed using HNE-modified synthetic peptides and also showing enrichment from a complex matrix of digested human plasma proteins. Applicability was confirmed using apomyoglobin as an analyte, implicating thereby its potential value to proteome-wide identification and relative quantification of posttranslational protein carbonylation with residue-specific information. Because HNE attachment may not necessarily cause change in protein abundance, this modification-focused quantification should facilitate the characterization of accompanied changes in protein function and, also, provide important insights into molecular signaling mechanisms and a better understanding of cellular processes associated with oxidative stress.
细胞膜脂质的过氧化作用，富含多不饱和脂肪酸，会产生亲电的、α，β-不饱和醛，如 4-羟基-2-壬烯醛（HNE）。HNE 是一种具有高反应性和细胞毒性的分子，可以与蛋白质中的亲核位点反应，导致翻译后修饰。鉴定蛋白质靶标是重要的第一步；然而，定量分析特定位置的修饰对于了解 HNE 诱导的羰基化对生物的影响是必要的。我们报告了一种使用轻（H(12)CHO）和重（D(13)CDO）同位素变体甲醛通过还原甲基化来差异标记肽中的伯胺（N 末端和赖氨酸的 ε-氨基）的方法，以及与修饰肽的选择性富集相结合，允许在混合后通过同时液相色谱-质谱对两种样品中羰基化的程度进行比较。具体而言，使用固相酰肼化学将二甲基标记的肽羰基从未修饰的肽中分离出来，以将其固定到多孔玻璃珠上，并在彻底洗涤珠子去除未修饰的肽后，通过酸催化水解来回收它们。该方法是使用 HNE 修饰的合成肽开发的，也显示出从消化的人血浆蛋白质的复杂基质中的富集。使用脱肌红蛋白作为分析物确认了适用性，从而暗示了其在蛋白质组范围内鉴定和相对定量翻译后蛋白质羰基化的潜在价值，具有残基特异性信息。由于 HNE 附着不一定会导致蛋白质丰度的变化，因此这种针对修饰的定量分析应该有助于描述伴随的蛋白质功能变化，并且还提供对分子信号机制的重要见解，并更好地了解与氧化应激相关的细胞过程。
