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Removal of the carbohydrate groups on a glycoprotein is recommended prior to protein identification. In general, glycoproteins and glycopeptides do not ionize
completely during MS analysis, leading to inadequate spectral data. Glycopeptides have lower
detection sensitivity due to microheterogeneity of the attached glycans, resulting in signal
suppression in MS analysis. Thus, removing the glycans increases protein sequence coverage for
protein identification. Furthermore, proteolytic cleavage is a prerequisite for the elution of
peptide fragments out of gels and identification by MS. However, proteolytic digestion of the
native glycoprotein is often incomplete due to steric hindrance from the bulky oligosaccharides present on the molecule. Information gained from MS analysis of deglycosylated tryptic peptides can be used to locate glycosylation sites on the glycoprotein.
The GlycoProfile™ In-Gel Enzymatic N-Deglycosylation Kit is optimized
to provide a convenient and reproducible method to N-deglycosylate and digest protein samples
from 1D or 2D polyacrylamide gel pieces for subsequent MS or HPLC analysis. The procedure is
suitable for Coomassie® Brilliant Blue and colloidal Coomassie stained gels. Silver
stained gels may also be used if properly destained.
The kit includes PNGase F and trypsin enzymes necessary for N-linked
deglycosylation and tryptic digestion, respectively. The samples can then be desalted and
concentrated for analysis by MALDI-TOF MS or Electrospray MS with subsequent database searching. |
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