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Comprehensive profiling and kinetic studies of glycated lysine residues in human serum albumin

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Abstract

Lysine residues of proteins slowly react with glucose forming Amadori products. In hyperglycemic conditions, such as diabetes mellitus, this non-enzymatic glycation becomes more pervasive causing severe medical complications. The structure and conformation of a protein predisposes lysine sites to differing reactivity influenced by their steric availability and amino acid microenvironment. The goal of our study was to identify these sites in albumin and measure glycation affinities of lysine residues. We applied a bottom-up approach utilizing a combination of three LC–MS instruments: timsTOF, Orbitrap, and QTRAP. To prove applicability to samples of varying glycemic status, we compared in vitro glycated and non-glycated HSA, as well as diabetic and non-diabetic individual samples. The analysis of lysine glycation affinities based on peptide intensities provide a semi-quantitative approach, as the results depend on the mass spectrometry platform used. We found that glycation levels based on multiple reaction monitoring (MRM) quantitation better reflect individual glycemic status and that the glycation percentage for each site is in linear relation to all other sites. To develop an approach which more accurately reflects glycation affinity, we developed a kinetics model which uses results from stable isotope dilution HPLC-MRM methodology. Through glycation of albumin at different glucose concentrations, we determine the rate constants of glycation for every lysine residue by simultaneous comparative analysis.

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Acknowledgements

We would like to thank Gehrke Proteomics Center and Molecular Interaction Core (University of Missouri, Columbia, USA) for peptide syntheses, CD experiments, and LC-MS acquisitions (timsTOF, Orbitrap).

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  1. Department of Pathology & Anatomical Sciences, School of Medicine, University of Missouri, 1 Hospital Drive, Columbia, MO, 65212, USA

    Aleks Shin, Yahor Vazmitsel, Shawn Connolly & Kuanysh Kabytaev

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Aleks Shin and Yahor Vazmitsel: investigation, data curation, writing—review and editing. Shawn Connolly: conceptualization, writing—review and editing. Kuanysh Kabytaev: conceptualization, methodology, writing—original draft, supervision.

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Correspondence to Kuanysh Kabytaev.

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Shin, A., Vazmitsel, Y., Connolly, S. et al. Comprehensive profiling and kinetic studies of glycated lysine residues in human serum albumin. Anal Bioanal Chem 414, 4861–4875 (2022). https://doi.org/10.1007/s00216-022-04108-1

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