Development of electrophoretic conditions for the characterization of protein glycoforms by capillary electrophoresis-electrospray mass spectrometry.
A capillary electrophoresis (CE) method using acidic buffers and capillaries coated with Polybrene, a cationic polymer has been developed for the separation of glycoproteins and glycopeptides. Electrophoretic conditions have been optimized to provide resolution of individual glycoforms observed for different glycoprotein preparations. These conditions were found to be entirely compatible with the operation of electrospray mass spectrometry (ESMS), which facilitated the assignments of possible carbohydrate compositions of glycopeptides arising from digests of glycoproteins. By using operating conditions enhanced the formation of oxonium fragment ions prior to mass spectral analysis, selective identification of glycopeptides was achieved for complex samples such as those from proteolytic digests or chemical cleavages. Examples of applications are presented for ribonuclease B, ovalbumin, horseradish peroxidase, and a lectin from Erithrina corallodendron using both CE-ESMS and CE with ultraviolet detection (CE-UV).
Kelly
J F
JF
Chemistry Department, Dalhousie University, Halifax, Nova Scotia, Canada.
Locke
S J
SJ
Ramaley
L
L
Thibault
P
P
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis*; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000032:analysis; D012260:Ribonucleases / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet
D006023:Glycoproteins; D010047:Ovalbumin; D012260:Ribonucleases