You need a database of oligosaccharide structures/masses, remember that the
glycopeptides don't "fly" too well so look for the small stuff in the spectra
and be prepared to spend lots of time looking at the data...it's there and it's
real-all you have to do is figure it out!!
Good luck! Jim
EXNETWRK.abrfreq@BAYERMAIL on 04/06/98 02:01:46 PM
To: "DDA.RFC-822=abrf@aecom.yu.edu/P=Internet/A= /C=us"@X400
cc:
Subject: CHO analysis by MS
I am new to CHO analysis on mass spec and am seeking advice from anyone who
has done this. I am collaborating with a group who have a protein that
looks to have a significant amount of glycosylation in 2-3 sites--there is
a 20,000 Da difference in mass on gels and predicted mass of protein
sequence. I've recovered all the peptides except three from in-gel
digests, two of them have predicted N-glycosylation sites. How good are
these prediction paradigms for finding glycosylation sites?
They are planning to use PNGase (N-glycanase) to see if it is
N-linked. In the discussion of CHO quantitation, someone mentioned that
there are many forms of O-glycanases--does that mean that each has a
different specificity, so you need to try several? If PNGase doesn't take
off the CHO, can you assume its O-linked? Is there a way to digest the
sugar down to one or two CHO units in order to identify the site on the
peptide?
Although the protein is not clean, it is the only glycosylated
protein in the sample. I wondered if I could digest the whole mixture of
proteins, then use some column to pull out the glycosylated peptides. I
was thinking of something like the column that is used to analyze
glycosylated hemoglobin. Can it be used for pulling CHO peptides out as
well?
My function in this collaboration is to provide what information I
can by mass spec analysis. Once I have the peptides out, what is the
recommended sequence of steps for analysis by mass spec, given that these
are likely to be fairly large CHO side chains? What reviews or references
on analysis of CHO would you recommend for me to start with (CHO analysis
is a bit overwhelming and requires new thought processes for someone used
to amino acids).
Thanks in advance
Katheryn Resing