Figure 2).

Results outlining temperature-based differences in glycopeptide identification. A) Higher concentration glycopeptide samples (i.e., the undiluted sample (1x), and first two serial dilutions (2x and 4x)) displayed the expected increase in identifications when separated at 45°C, stemming largely from accessing new glycoprotein constituents. Concentration and temperature are independent; all concentrations (i.e., dilutions) were analyzed at all temperatures. B) Our analyses showed high technical reproducibility indicating the changes in unique glycosites between temperature is unlikely due to analytical abnormalities. C) The peptide backbone information of glycopeptides was largely conserved across temperatures, suggesting major differences are not due to the backbone itself. D) Glycopeptides demonstrated a class-dependent response to temperature with mannose type glycans benefitting from higher temperatures; complex, fucosylated and sialylated types show decreased identifications at high temperatures.