Identification of heavily glycated proteoforms by hydrophilic-interaction liquid chromatography and native size-exclusion chromatography - High-resolution mass spectrometry

In this study, we investigated two novel LC-HRMS methods to study glycated reference proteins at the intact protein level: low-flow hydrophilic-interaction liquid chromatography (HILIC) and native size-exclusion chromatography (SEC). Model proteins were exposed to conditions that favored extensive glycation and the formation of AGEs. After glycation, complicated MS spectra were observed, along with a sharply reduced signal response, possibly due to protein denaturation and the formation of aggregates. When using HILIC-MS, the glycated forms of the proteins could be resolved based on the number of reducing monosaccharides. Moreover, some positional glycated isomers were separated. The SEC-MS method under non-denaturing conditions provided insights into glycated aggregates but offered only a limited separation of glycated species based on molar mass. Overall, more than 25 different types of species were observed in both methods, differing in molar mass by 14-162 Da. 19 of these species have not been previously reported.SIGNIFICANCE: The proposed strategies show great potential to characterize highly glycated intact proteins from native and denaturing perspectives and provide new opportunities for fast clinical diagnoses and investigating glycation-related diseases.PMID:38637052 | DOI:10.1016/j.aca.2024.342543

Source: Analytica Chimica Acta - April 18, 2024 Category: Chemistry Authors: Ziran Zhai Peter J Schoenmakers Andrea F G Gargano Source Type: research