separation of tryptic digestion

kzhao at BIOVX1.BIOLOGY.UCLA.EDU kzhao at BIOVX1.BIOLOGY.UCLA.EDU
Mon Oct 28 13:29:56 EST 1996


A message from e8327091 at stud1.tuwien.ac.at asked the following question:

Dear Glycoscientists!
I am searching for an easy (like always) method to separate
peptides from glycopeptides.
After a tryptic digest and RP chromatography, I would like to identify
glycopeptides. Because of the complex peak pattern of the RP map
it is unpossible to use a PNGase digest after the tryptic digest
and compare or fractionate the RP maps.
A possibility would be the use of a ConA column, but I think that the
ConA material is too specific.
I would prefer a HPLC method, because there is only a mg amount of material.
So if anyone of you has suggestions or experience with this problem, please
post to the newsgroup, or mail to e8327091 at stud1.tuwien.ac.at.
Thanks a lot
                Martin Kaliwoda 


My experience on glycopeptide analysis tells me that it is very difficult to
analyze glycopeptides among non-glycosylated ones after tryptic digestion.

It's relative ideal to use a series of lectin columns starting from Con A, 
followed by wheat germ agglutinin, and PHA, or other combinations depending
on your particular case.  HPLC separation of lectin eluted fractions will
give you a quite clean peaks.  You also have to bear in mind that trypsin
may not give you all the necessary glycopeptides you had expected even you
denatured and carboxymethylated your protein.  It surprised me that you have
to use "only" a mg amount of material.  I merely used about 0.2 mg of a
80 kD glycoprotein to do both tryptic and chymotryptic digestions and obtained
desired information.

********************************************************************************
* Ke-Wei Zhao, Ph.D.                     Phone: (310)825-8722                  *
* Department of Biological Chemistry     Fax: (310)206-5272                    *
* UCLA School of Medicine, CHS 33-257    e-mail: kzhao at biovx1.biology.ucla.edu *
* Los Angeles, CA 90095-1737                                                   *
********************************************************************************




More information about the Glycosci mailing list