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Observational Study
. 2020 Apr 15;10(1):6475.
doi: 10.1038/s41598-020-62341-z.

Serum albumin cysteine trioxidation is a potential oxidative stress biomarker of type 2 diabetes mellitus

Selvam Paramasivan  1 , Sunil S Adav  1 , SoFong Cam Ngan  1 , Rinkoo Dalan  2 , Melvin Khee-Shing Leow  2   3 , Hee Hwa Ho  4 , Siu Kwan Sze  5
Affiliations
Observational Study

Serum albumin cysteine trioxidation is a potential oxidative stress biomarker of type 2 diabetes mellitus

Selvam Paramasivan et al. Sci Rep. .

Abstract

Metabolic disorders in T2DM generate multiple sources of free radicals and oxidative stress that accelerate nonenzymatic degenerative protein modifications (DPMs) such as protein oxidation, disrupt redox signaling and physiological function, and remain a major risk factor for clinical diabetic vascular complications. In order to identify potential oxidative biomarkers in the blood plasma of patients with T2DM, we used LC-MS/MS-based proteomics to profile plasma samples from patients with T2DM and healthy controls. The results showed that human serum albumin (HSA) is damaged by irreversible cysteine trioxidation, which can be a potential oxidative stress biomarker for the early diagnosis of T2DM. The quantitative detection of site-specific thiol trioxidation is technically challenging; thus, we developed a sensitive and selective LC-MS/MS workflow that has been used to discover and quantify three unique thiol-trioxidized HSA peptides, ALVLIAFAQYLQQC(SO3H)PFEDHVK (m/z 1241.13), YIC(SO3H)ENQDSISSK (m/z 717.80) and RPC(SO3H)FSALEVDETYVPK (m/z 951.45), in 16 individual samples of healthy controls (n = 8) and individuals with diabetes (n = 8). Targeted quantitative analysis using multiple reaction monitoring mass spectrometry revealed impairment of the peptides with m/z 1241.13, m/z 717.80 and m/z 951.45, with significance (P < 0.02, P < 0.002 and P < 0.03), in individuals with diabetes. The results demonstrated that a set of three HSA thiol-trioxidized peptides, which are irreversibly oxidatively damaged in HSA in the plasma of patients with T2DM, can be important indicators and potential biomarkers of oxidative stress in T2DM.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic workflow showing three-phase experiments designed to identify, validate and quantify cysteine trioxidation. In the discovery phase, pooled plasma samples were subjected to proteomics profiling using data-dependent LC-MS/MS. Human serum albumin (HSA) was identified with significant Cys trioxidation. In phase II (HSA targeted phase), plasma samples were separated by SDS-PAGE, and albumin bands were excised for in-gel digestion and analyzed by LC-MS/MS to confirm the discovery phase results. In the quantitation phase, three HSA Cys-trioxidized peptides and their unmodified counter peptide pairs with strong signals were quantitatively measured using the MRM targeted proteomic method. The three peptide pairs were also synthesized and measured by both data-dependent MS/MS and MRM methods to confirm the positive identification of the trioxidation sites. The validated MRM method was used to quantitatively study HSA Cys trioxidation in plasma samples from patients with T2DM and healthy controls.
Figure 2
Figure 2
MS/MS spectrum of peptide ALVLIAFAQYLQQCPFEDHVK showing (A) trioxidation and (B) carbamidomethylation along with their fragmentation profiles. The Skyline dotp values of Cys34 trioxidation and unmodified counter peptides were 0.87 and 0.92, respectively, indicating high confidence identification and MRM quantitation of the peptides.
Figure 3
Figure 3
MRM MS/MS spectra, fragmentation profiles and Skyline dpot values of HSA Cys-trioxidized peptide, YIC(SO3H)ENQDSISSK. (A) Peptide identified in the plasma of patients with T2DM. (B) Results from synthetic peptide showing identical MS/MS fragmentation profile as the tryptic peptide from endogenous HSA.
Figure 4
Figure 4
MRM MS/MS spectra, fragmentation profiles and Skyline dotp values of HSA Cys-trioxidized peptide, RPC(SO3H)FSALEVDETYVPK. (A) Peptide identified in the plasma of patients with T2DM. (B) Results from synthetic peptide showing identical MS/MS fragmentation profile as the tryptic peptide from endogenous HSA.
Figure 5
Figure 5
Retention time of LC separation of trioxidized and carbamidomethylated peptides. The unmodified (cysteine carbamidomethylation) and cysteine trioxidation peptides (in both A) RPCFSALEVDETYVPK and (B) YICENQDSISSK peptides) were well separated by reverse phase liquid chromatography using a C18 column. The retention times of the plasma tryptic peptides and the corresponding synthetic peptides were similar in the data-dependent LC-MS/MS. These retention times were then applied to the instrumental settings of the scheduled LC-MRM-MS. The variation of the retention time of the same peptide was less than 1 minute across 16 plasma samples.
Figure 6
Figure 6
Quantitative cysteine trioxidation at different sites. Statistical significance is analyzed by one-way ANOVA.
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