title: Sample matrix effects on glycopeptide stability by high performance capillary electrophoresis.
abstract: High performance capillary electrophoresis (CE) of glycoprotein digests frequently reveals extensive microheterogeneity associated with specific protein glycosylation sites. The choice of the sample matrix can influence the electrophoretic migration time, peak shape and resolution, as well as the physical stability of the product glycopeptides. Acetic acid is a frequently employed sample matrix for both capillary electrophoresis and electrospray ionization-mass spectrometry (ESI-MS). Acetic acid appears to enhance the spontaneous hydrolysis of sialic acids from the nonreducing termini of glycopeptides in a time- and concentration-dependent manner, even at 5 degrees C, as evidenced by changes in the electrophoretic mobility and ESI-MS spectra of the resulting glycopeptides. The observed parallel electrophoretic mobility changes for specific glycoforms are consistent with the induction of peptide structure with time. Asialoglycopeptide mobilities were stable in acetic acid. Electrophoretic mobilities can be stabilized with propionic acid 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 enhanced glycopeptide resolution.
authors.affiliation: Department of Protein Structure, Amgen Inc., Thousand Oaks, CA 91320-1789, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: Rush
authors.forename: H J
authors.initials: HJ
authors.lastname: Boss
authors.forename: V
authors.initials: V
authors.lastname: Katta
authors.forename: M F
authors.initials: MF
authors.lastname: Rohde
mesh_terms: D000085:Acetates / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D011422:Propionates / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000085:Acetates; D006020:Glycopeptides; D011422:Propionates; D011994:Recombinant Proteins; D004921:Erythropoietin