In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.
Kilz
S
S
Institut für Organische Chemie der Universität zu Köln, Greinstrasse 4, 50939 Cologne, Germany.
Budzikiewicz
H
H
Waffenschmidt
S
S
D000818:Animals; D002241:Carbohydrates / Q000032:analysis* / Q000302:isolation & purification; D002696:Chlamydomonas / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D006909:Hydroxyproline / Q000737:chemistry; D010084:Oxidation-Reduction; D011725:Pyridines / Q000032:analysis; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002241:Carbohydrates; D006023:Glycoproteins; D011725:Pyridines; D006909:Hydroxyproline