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

## TL;DR

This study uses advanced mass spectrometry to identify and quantify protein carbonylation in human serum, revealing common modification sites and suggesting these changes are not specific to rheumatoid arthritis.

## Contribution

A novel strategy combining ARP derivatization, IMS, and DIA for high-throughput detection of protein carbonylation in serum.

## Key findings

- 86 ARP-peptides were manually confirmed with high signal-to-background ratios.
- 28 carbonylation sites were identified on human serum albumin, including several hotspots.
- Six previously unreported carbonylation species were detected using advanced MS techniques.

## 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.