Biology Reference
In-Depth Information
4
Notes
1. Phenylmethanesulfonylfl uoride (PMSF) can be added to the
protease inhibitor cocktail (1 mM fi nal concentration) for better
protease inhibition.
2. Is important to note that cartridges used for the fi rst time can
leak some of the lectin during the glycoprotein elution step.
Therefore, it is recommended to wash and equilibrate the
columns with more than 10 volumes of binding buffer prior to
application of the sample.
3. When the volume of the sample is large and the proteins
concentration is low, it may help to perform the loading step in
a cold room with a peristaltic pump. It is important to load the
samples at as a low a rate as possible. Alternatively, a fast protein
liquid chromatography (FPLC; GE Healthcare) system with a
sample loop can be used.
4. Protein samples in buffers containing urea must not be heated
because this can cause carbamylation: urea in solution is in equi-
librium with ammonium cyanate and the isocyanic acid reacts
with protein amino groups. This results in considerable charge
heterogeneity, which complicates subsequent MS analysis.
5. The majority of the glycopeptides bind to the PGC matrix;
however, some peptides and glycopeptide do not bind. If an
objective is to identify non-glycosylated peptides that were
derived from the original glycoproteins, then analysis of the
fl ow through may also be included at this point.
Acknowledgments
Funding to JKCR for research in this area is provided by the NSF
Plant Genome Research Program (DBI-0606595) and the New
York State Offi ce of Science, Technology and Academic Research
(NYSTAR).
References
1. Marino K, Bones J, Kattla JJ et al (2010) A
systematic approach to protein glycosylation
analysis: a path through the maze. Nat Chem
Biol 6:713-723
2. An HJ, Froehlich JW, Lebrilla CB (2009)
Determination of glycosylation sites and
site-specifi c heterogeneity in glycoproteins.
Curr Opin Chem Biol 13:421-426
3. Pless DD, Lennarz WJ (1977) Enzymatic con-
version of proteins to glycoproteins. Proc Natl
Acad Sci USA 74:134-138
4. Matsuoka K, Watanabe N, Nakamura K (1995)
O- glycosylation of a precursor to a sweet
potato vacuolar protein, sporamin, expressed
in tobacco cells. Plant J 8:877-889
5. Cho YP, Chrispeels MJ (1976) Serine-
O-galactosyl linkages in glycopeptides
from carrot cell-walls. Phytochemistry 15:
165-169
6. Showalter AM (2001) Arabinogalactan-proteins:
structure, expression and function. Cell Mol Life
Sci 58:1399-1417
 
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