SAMPLE MATRIX EFFECTS ON GLYCOPEPTIDE STABILITY BY HIGH-PERFORMANCE CAPILLARY ELECTROPHORESIS

High performance capillary electrophoresis (CE) of glycoprotein digest s frequently reveals extensive microheterogeneity associated with spec ific protein glycosylation sites. The choice of the sample matrix can influence the electrophoretic migration rime, peak shape and resolutio n, as well as the physical stability of the product glycopeptides. Ace tic acid is a frequently employed sample matrix for both capillary ele ctrophoresis and electrospray ionization-mass spectrometry (ESI-MS). A cetic acid appears to enhance the spontaneous hydrolysis of sialic aci ds from the nonreducing termini of glycopeptides in a time- and concen tration-dependent manner, even at 5 degrees C, as evidenced by changes in the electrophoretic mobility and ESI-MS spectra of the resulting g lycopeptides. The observed parallel electrophoretic mobility changes f or specific glycoforms are consistent with the induction of peptide st ructure with time. Asialoglycopeptide mobilities were stable in acetic acid. Electrophoretic mobilities can be stabilized with propionic aci d sample matrix with no apparent structural changes observed by ESI-MS within 31 h. Migration time reproducibility was in the range of 0.1% relative standard deviation (N = 7) with excellent peak shapes and enh anced glycopeptide resolution.