"Sequencing of N-Linked Oligosaccharides Directly from Protein Gels: In-Gel
Deglycosylation Followed by Matrix-Assisted Laser Desorption/Ionization
Mass Spectrometry and Normal-Phase High-Performance Liquid Chromatography"
Kuster et al., Analytical Biochemistry 250, 82-101 (1997)
-Young Moo
You wrote:
>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
____
Young Moo Lee, Ph.D.
Director
Protein Structure Laboratory
University of California
Davis, California 95616-8656
530-752-4328 (voice)
530-752-8969 (fax)
ymlee@ucdavis.edu
http://pubweb.ucdavis.edu/Documents/psl/psl_pg.htm