Abstract
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.