Proteome-wide Detection and Quantitative Analysis of Irreversible Cysteine Oxidation Using Long Column UPLC-pSRM

Reactive
oxygen species (ROS) play an important role in normal
biological functions and pathological processes. ROS is one of the
driving forces for oxidizing proteins, especially on cysteine thiols.
The labile, transient, and dynamic nature of oxidative modifications
poses enormous technical challenges for both accurate modification
site determination and quantitation of cysteine thiols. The present
study describes a mass spectrometry-based approach that allows effective
discovery and quantification of irreversible cysteine modifications.
The utilization of a long reverse phase column provides high-resolution
chromatography to separate different forms of modified cysteine thiols
from protein complexes or cell lysates. This Fourier transform mass
spectrometry (FT-MS) approach enabled detection and quantitation of
ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation
states using Skyline MS1 filtering. When we applied the long column
ultra high pressure liquid chromatography (UPLC)–MS/MS analysis,
61 and 44 peptides from cell lysates and cells were identified with
cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure
liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM)
was then developed to monitor the oxidative level of cysteine thiols
in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion
of sulfinic (S-O2H) and sulfonic acid (S-O3H)
was observed within nucleoside diphosphate kinase (Nm23-H1) and heat
shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide
studies, providing a highly sensitive, straightforward approach to
identify proteins containing redox-sensitive cysteine thiols in biological
systems.

活性氧簇（Reactive oxygen species, ROS）在正常生理功能与病理进程中发挥关键作用。ROS是氧化蛋白质的核心驱动力之一，尤其针对半胱氨酸巯基。氧化修饰具有不稳定、瞬时且动态的特性，这对半胱氨酸巯基的精准修饰位点鉴定与定量分析均带来了极大的技术挑战。本研究报道了一种基于质谱法的研究策略，可有效实现半胱氨酸不可逆修饰的发现与定量分析。长反相色谱柱可提供高分辨率色谱分离能力，能够从蛋白质复合物或细胞裂解液中分离不同形式的修饰型半胱氨酸巯基。本研究采用的傅里叶变换质谱（Fourier transform mass spectrometry, FT-MS）策略，借助Skyline MS1过滤技术，实现了共济失调毛细血管扩张症突变（ataxia telangiectasia mutated, ATM）复合物半胱氨酸亚磺酰化状态的检测与定量。当应用长柱超高效液相色谱-串联质谱（ultra high pressure liquid chromatography–MS/MS, UPLC-MS/MS）分析时，分别在体外与体内过氧化氢氧化处理的细胞裂解液与细胞样本中，鉴定得到61条与44条带有半胱氨酸修饰的肽段。随后，研究人员开发了长柱超高效液相色谱伪选择反应监测（ultra high pressure liquid chromatography pseudo selected reaction monitoring, UPLC-pSRM）方法，用于监测不同浓度过氧化氢处理下细胞裂解液中半胱氨酸巯基的氧化水平。通过UPLC-pSRM分析，研究人员在核苷二磷酸激酶（nucleoside diphosphate kinase, Nm23-H1）与热休克70kDa蛋白8（heat shock 70 kDa protein 8, Hsc70）中观测到了亚磺酰基（S-O₂H）与磺酰基（S-O₃H）的动态转化过程。上述方法适用于全蛋白质组学研究，可为生物系统中含氧化还原敏感型半胱氨酸巯基的蛋白质鉴定提供高灵敏度、简便易行的技术路径。