Identification of Heavily Glycated Proteoforms by Hydrophilic-Interaction Liquid Chromatography and Native Size-Exclusion Chromatography – High-Resolution Mass Spectrometry

The non-enzymatic glycation of proteins and their advanced glycation end products (AGEs) are associated with protein transformations such as in the development of diseases and biopharmaceutical storage. The high heterogeneity that glycated proteins carry makes their detection and identification challenging. 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). Three model proteins (RNase-A, hemoglobin, and NISTmab) 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 to 162 Da. 19 of these species have not been previously reported. By tracing the progress of glycation, the dynamic changes of the specific AGEs could be monitored over time.