Detection of highly hydrophilic glycopeptides in reverse-phase liquid chromatography-mass spectrometry after TMTPro Zero labeling

Bottom-up analysis of glycopeptides by liquid chromatography-mass spectrometry (LC-MS) plays a critical role in the detection and relative quantification of all glycoforms at each glycosylation site. Reversed-phase (RP) nanoLC-MS in particular is a favored technique for analyzing highly glycosylated proteins due to its sensitivity and accessibility. However, analyzing glycopeptides with large, diverse glycan chains presents challenges such as insufficient and largely varying chromatographic retention. Specifically, hydrophilic glycopeptides consisting of a short, hydrophilic peptide portion with a large, hydrophilic glycan are not retained. We present a RP-nanoLC-MS method that allows for the full characterization of highly hydrophilic glycopeptides. Glycopeptide retention was increased using the amine-reactive and relatively hydrophobic TMTPro Zero label that was covalently attached to all (glyco)peptides. To further increase retention, endoproteinase Lys-C was used to perform proteolysis, generating glycopeptides exhibiting two primary amine groups that were labeled with two TMTPro Zero tags, thereby achieving RP retention of the most hydrophilic glycopeptide analytes. The workflow was applied to two E. coli O-antigen bioconjugates exhibiting highly diverse N-glycopeptides with glycan chains ranging in size between 4 and more than 70 monosaccharides. This optimized approach enables bottom-up analysis of glycoproteins exhibiting large glycans of very high hydrophilicity, supporting comprehensive site-specific glycosylation analysis. HYPOTHESIS: Different glycopeptides with unfavorable chromatographic properties can be detected and characterized using TMTPro Zero labeling and mass spectrometry.