Monitoring protein glycation by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) mass spectrometer

• Extent of glycation was monitored by ESI-QTOF-MS technique in different conditions.
• Heating with glucose in solution resulted in one mol glucose attachment to proteins.
• Multiple glycoforms were formed by heating proteins with glucose in dry state.
• ESI-QTOF-MS technique allowed a fast evaluation of early glycation of proteins.

In this study electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry was used to investigate protein glycation. The glycated species of cytochrome C, lysozyme, and β-casein formed during glycation with d-glucose were identified and monitored in binary systems heated at 70 °C under dry and aqueous conditions. Cytochrome C had multiple charges in non-glycated state, primarily changing from +13 to +17 positive charges, whereas β-casein had charge states up to +30. Upon heating with glucose at 70 °C in aqueous state, attachment of one glucose molecule onto proteins was observed in each charge state. However, heating in dry state caused much more glucose attachment, leading to the formation of multiple glycoforms of proteins. By using ESI-QTOF-MS technique, formation of glycated cytochrome C containing up to 12 glucose moieties were observed, while glycated species containing 6 and 8 glucose moieties were observed for lysozyme and β-casein, respectively in various heating conditions.