Screening of Protein Carbonylation Sites in Human Serum by Ion Mobility Mass Spectrometry

Excessive oxidative
stress, associated with various diseases, can
induce protein carbonylation-nonenzymatic modifications involving
aldehyde or keto group formation. These modifications are structurally
diverse and low in abundance, which complicates their detection and
quantitation. Here, we developed a strategy to identify and quantify
protein carbonylation in human serum proteins from 39 rheumatoid arthritis
patients and 29 healthy donors. Reactive carbonyl groups were derivatized
with an aldehyde reactive probe (ARP), digested with trypsin, enriched
via avidin affinity chromatography, and analyzed using RP-HPLC-ESI-IMS-MS/MS.
Ion mobility spectrometry (IMS) was applied in both data-dependent
(DDA) and data-independent acquisition (DIA) modes. DDA generated
spectral libraries of ARP-derivatized peptides (ARP-peptides), which
enabled peptide-centric detection in DIA data. We manually confirmed
86 ARP-peptides, with 93.8% of peak areas showing signal-to-background
ratios >3. Among the 32 unique carbonylation sites, 28 were on
human
serum albumin, with hotspots at Cys58, Lys214, Lys219, Lys223, Lys456,
Lys543, Lys549, and Lys565. Six previously unreported species were
identified using IMS, DIA, ARP-reporter ions, and de novo sequencing. The ARP-peptides were quantified with ≥ 75% intrabatch
reproducibility (coefficient of variation <20%). Similar modification
levels were observed in both groups, suggesting basal, disease-independent
carbonylation in abundant serum proteins.

过度氧化应激与多种疾病密切相关，可诱发蛋白质羰基化——即涉及醛基或酮基形成的非酶促修饰反应。这类修饰结构多样且丰度较低，给其检测与定量分析带来了极大挑战。本研究开发了一套分析策略，用于对39名类风湿关节炎（rheumatoid arthritis）患者与29名健康志愿者的人血清蛋白质中的羰基化修饰进行鉴定与定量。研究中将活性羰基基团与醛反应探针（aldehyde reactive probe, ARP）进行衍生化处理，随后经胰蛋白酶（trypsin）酶解、亲和素（avidin）亲和色谱富集，并采用反相高效液相色谱-电喷雾电离-离子迁移谱-串联质谱（RP-HPLC-ESI-IMS-MS/MS）进行分析。本研究采用了数据依赖型采集（data-dependent acquisition, DDA）与数据非依赖型采集（data-independent acquisition, DIA）两种模式开展离子迁移谱（Ion mobility spectrometry, IMS）分析。DDA模式可构建ARP衍生肽（ARP-peptides）的光谱库，从而实现DIA数据中以肽段为中心的检测分析。本研究手动验证了86条ARP衍生肽，其中93.8%的峰面积信噪比大于3。在32个独特的羰基化修饰位点中，有28个位于人血清白蛋白（human serum albumin）上，其修饰热点为Cys58、Lys214、Lys219、Lys223、Lys456、Lys543、Lys549与Lys565。本研究结合IMS、DIA、ARP报告离子与从头测序（de novo sequencing）技术，鉴定出6种此前未被报道的修饰形式。ARP衍生肽的定量批内重复性≥75%（变异系数<20%）。两组受试者的修饰水平无显著差异，提示丰度较高的血清蛋白存在不依赖于疾病状态的基础羰基化修饰。