We have tried the LC-MS/MS method to directly analyse glycopeptides from
tryptic digested recombinant lysosomal alpha-L-iduronidase which pocesses
6 utilized N-glycosylation sites. The danger of this technique is that
you have no chance to verify the actual modification merely based on the
total masses. We later switched to off-line MALDI-TOFMS from lectin
purified glycopeptides which were then cleaned on HPLC. This procedure
led us unambiguously identified almost all of the 6 oligosaccharides as
well as biphosphomonoesters on two of the four high mannose structures.
Since 2 phosphates (~160 Da) and 2 sulfates (also ~160 Da) are not
different from a hexose (162.14 Da), it is very important that proper
enzymes be used to verify your moiety of interest. In my case, I can
digest the phosphorylated peak with E. coli alkaline phosphatase, and
at the same time,two new peaks, 80 and 160 Da smaller, respectively, were
seen. This can hardly be done without lectin purification and without
enzyme digestion and MALDI-TOFMS re-assay.
Therefore, depending on how many sugar chains on your glycoprotein, you
should determine which strategies are more suitable for your purpose.
What I found unexpectedly in my MALDI-TOFMS, that trypsin (TPCK-treated)
does not always cleave at the expected K or R (except followed by P).
This added more complications to LC-MS/MS.
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*Ke-Wei Zhao, Ph.D.
*Dept. of Biological Chemistry
*UCLA School of Medicine
*Los Angeles, CA 90095-1737
*(310)825-8722 (Tel)
*(310)206-5272 (Fax)
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