Steckel Arnold, Uray Katalin, Turiák Lilla, Gömöry Ágnes, Drahos László, Hudecz Ferenc, Schlosser Gitta
Doctoral School of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary.
MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, ELTE Eötvös Loránd University, Budapest, Hungary.
Rapid Commun Mass Spectrom. 2018 Jun 15;32(11):844-850. doi: 10.1002/rcm.8105.
Protein citrullination (deimination) is a post-translational modification of proteins converting arginine(s) into citrulline(s). "Overcitrullination" could be associated with severe pathological conditions. Mass spectrometric analysis of modified proteins is hindered by several problems. A comprehensive study of the fragmentation of deiminated peptides is not yet available. In this paper we have made an attempt to describe the characteristics of these processes, based on the studies of epitope model oligopeptides derived from clinically relevant proteins.
Solutions of purified model peptides containing either one or two citrulline residues as well as their native variants were injected directly into the electrospray source of a high accuracy and resolution quadrupole-time-of-flight instrument and were analysed by tandem mass spectrometry using low-energy collision-induced dissociation.
Loss of isocyanic acid from citrulline residues is a preferred fragmentation route for deiminated peptides, which yields ornithine residues in the sequence. However, simultaneous detection of both the isocyanic acid loss and sequence fragments is often compromised. A preferential cleavage site was observed between citrulline and any other following amino acids yielding intensive complementary b- and y-type ions. Also, citrulline positioned at the C-termini displays a preferential cleavage N-terminal to this residue yielding characteristic y ions. These phenomena are described here for the first time and are referred to as the "citrulline effect".
We found that the citrulline effect is very pronounced and could be used as a complementary tool for the confirmation of modification sites in addition to losses of isocyanic acids from the protonated molecules or from fragment ions. Low collision energy applied to peptide ions having partially mobile protons reveals the site of modification by generating specific and intensive fragments of the sequence. On the other hand, fragmenting precursor ions with mobile protons usually allows full sequence coverage, although citrulline-specific fragments may exhibit lower intensities compared to other fragments.
蛋白质瓜氨酸化（脱氨）是一种蛋白质的翻译后修饰，可将精氨酸转化为瓜氨酸。“过度瓜氨酸化”可能与严重的病理状况相关。修饰蛋白质的质谱分析存在若干问题。目前尚未有对脱氨肽片段化的全面研究。在本文中，我们基于对源自临床相关蛋白质的表位模型寡肽的研究，尝试描述这些过程的特征。
将含有一个或两个瓜氨酸残基的纯化模型肽溶液及其天然变体直接注入高精度和分辨率的四极杆-飞行时间仪器的电喷雾源，并使用低能碰撞诱导解离通过串联质谱进行分析。
瓜氨酸残基中异氰酸的丢失是脱氨肽的首选片段化途径，该途径会在序列中产生鸟氨酸残基。然而，异氰酸丢失和序列片段的同时检测常常受到影响。在瓜氨酸与任何后续氨基酸之间观察到一个优先切割位点，可产生强烈的互补b型和y型离子。此外，位于C末端的瓜氨酸在该残基的N末端显示出优先切割，产生特征性的y离子。这些现象首次在此描述，并被称为“瓜氨酸效应”。
我们发现瓜氨酸效应非常显著，除了质子化分子或碎片离子中异氰酸的丢失外，还可作为确认修饰位点的补充工具。对具有部分移动质子的肽离子施加低碰撞能量，通过产生序列的特定且强烈的片段来揭示修饰位点。另一方面，对具有移动质子的前体离子进行碎片化通常可实现完整的序列覆盖，尽管与其他片段相比，瓜氨酸特异性片段的强度可能较低。
