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

Abstract

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.

The Chaotrope-soluble Glycoprotein GP2 Is a Precursor of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall

“…3, lane 2) could be identified in the mixture of polypeptides released from the insoluble cell wall fraction by treatment with HF/pyridine (Fig. 1B), with the apparent exception of the 65-kDa deglycosylation product of GP3A (33). The molecular mass of deglycosylated GP3A corresponds roughly to that of the prominent 64-kDa component found in the polypeptides released from the insoluble wall fraction by treatment with HF/pyridine (Fig.…”

“…However, these 14 putatively GP2-derived peptides cover only 15% of this particular cell wall polypeptide. This is obviously due to the fact that GP2 is highly glycosylated (33). Furthermore, the experimental proof of its presence in the insoluble glycoprotein framework of the Chlamydomonas cell wall is hindered by cross-linking via intermolecularisodityrosineformation(6),transglutaminasedependent reactions (7), or carbohydrate side chains (32).…”

“…Both domains form the 100-nm long fibrous structure of this molecule (21,31), whereas the globular head (21) can be attributed to the C-terminal domain (domain 4). MALDI-TOF analyses of native GP2 and chemically deglycosylated GP2 revealed molecular masses of 281,250 and 142,600 Da, respectively, indicating a carbohydrate content of 49% (33). A putative partial amino acid sequence derived from the nucleotide sequence of a cloned gene fragment was published by Adair and Apt (34).…”

“…A putative partial amino acid sequence derived from the nucleotide sequence of a cloned gene fragment was published by Adair and Apt (34). GP3 is a disulfide-linked heterodimer (21,33). MALDI-TOF analyses of GP3A and GP3B revealed molecular masses of 131,500 and 144,400 Da, respectively (33).…”

“…GP3 is a disulfide-linked heterodimer (21,33). MALDI-TOF analyses of GP3A and GP3B revealed molecular masses of 131,500 and 144,400 Da, respectively (33). After chemical deglycosylation, molecular masses of 70,550 and 102,290 Da were found for GP3A and GP3B (33), respectively, indicating that the carbohydrate content of GP3A is considerably higher than that of GP3B.…”