Matrix-assisted laser desorption/ionization on-target method for the investigation of oligosaccharides and glycosylation sites in glycopeptides and glycoproteins.
The significance of glycoproteins in living systems instigates the ceaseless expansion of new techniques and procedures for the analysis of biological samples. Many of these applications are focused on improving the detection limit of analyzed material. In a previous study, we described a procedure for the detection of oligosaccharides cleaved from tryptic glycopeptides. Treatment of deglycosylated fractions with phenylhydrazine gave rise to peaks consistent with labeled glycans, and both types of compounds--deglycosylated peptides and oligosaccharides--were recorded from one spot and observed in one matrix-assisted laser desorption/ionization (MALDI) mass spectrum for the first time. Here, we added an additional step to this simple procedure of deglycosylating glycopeptides directly from the target spot of the first analyzed glycosylated peptides. For the purpose of this new study, a mixture of 2-aza-2-thiothymine and phenylhydrazine hydrochloride showed to be an excellent matrix for glycopeptides, oligosaccharides, deglycosylated peptides and moreover it allowed PNGaseF to be active enough to cleave oligosaccharides from peptides. The efficiency of this procedure is demonstrated on a series of intact glycoproteins and on the analysis of tryptic peptides obtained from IgG and total mouse serum. This one-step on-target deglycosylation method with subsequent derivatization on the same spot makes MALDI-MS analyses of glycopeptides fast, simple and accessible for biological samples, where classical procedures cannot produce useful results.
Lattová
Erika
E
Chemistry Department, University of Manitoba, Winnipeg, MB, Canada R3T 2N2. lattovae@cc.umanitoba.ca
Chen
Vincent C
VC
Varma
Sonal
S
Bezabeh
Tedros
T
Perreault
Hélène
H
D002241:Carbohydrates / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis*; D006031:Glycosylation; D006868:Hydrolysis; D007202:Indicators and Reagents; D009844:Oligosaccharides / Q000032:analysis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D002241:Carbohydrates; D006020:Glycopeptides; D006023:Glycoproteins; D007202:Indicators and Reagents; D009844:Oligosaccharides; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase