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