Re: MS of MAPs

Rachel Loo (ogorzloo@umich.edu)
Thu, 23 Apr 1998 08:43:13 -0400

Hi Ken and Manfred,
We had the poster on MAPs next to Manfred's at the 1994 ASMS
conference, mostly
by electrospray MS analysis, some MALDI. At the time, we were mostly
interested in
investigating charging in ESI-MS, but were also curious about the quality
of MAP spectra in general. We also got mostly ugly spectra, with the
occasional pretty one. Phil Andrews and I considered either small amounts
of sequence heterogeneity in different locations on the peptide chains,
(which would be hard to
pick up by Edman sequencing or amino acid analysis) or the higher order
structure of
the MAPs leading to aggregation or water retention or other problems. For
example,
for an 8-branch MAP missing a leucine at one position on one chain, the
small amount of preview observed in Edman sequencing might make the loss
undetectable. Add to this, incomplete removal of side-chain protecting
groups or migration to neighboring residues,
as well as oxidation, aspartimide bond formation, etc. and it gets messy
quickly. All of
these problems are likely to be partial on individual chains, making it
possible to
sequence and see mostly the right residue at each cycle. To address these
issues, we tried
ESI-MS under a range of denaturing conditions and temperatures and the ugly
ducklings
never turned into swans. For "resolution-challenged" spectra, where we got
broad peaks,
we could calculate a rough mass, always low. We synthesized many short test
MAPs, did MS,then cleaned them up by HPLC and reanalyzed. They behaved
fine by MS after HPLC and so it seemed to us that the ducks were ugly
becaused of how they hatched, and not by our perceptions.

Philip Andrews and Rachel Ogorzalek Loo
University of Michigan
2560 MSRB II
Ann Arbor, MI 48109-0674
(734) 647-0951
andrewsp@umich.edu ogorzloo@umich.edu

Hallo Ken,
we spent some time in the analysis of MAPS by ESI-MS and also (but very
short time) by MALDI-MS. I had a poster on the Chicago ASMS, directly
beside another poster about the same question and we had similar
experiences. Some MAPS work like linear peptides, a substance-P MAP looked
very nice, other show only broad peaks with no clear visible mass while
other didnot show anything, in ESI-MS. The same for MALDI-MS. We sequenced
the MAPS and could not see any mistakes in sequences, in most cases I could
reach the starting Ala. As far as one could say from the sequence analysis
all MAPS were fine. We discussed the reasons for the mass spectra and could
not find a final answer. I had the idea that due to the structure some MAPS
may carry water with them and do not loose the water in the spray and this
causes the broad peaks. So we thought about the SCIEX turbo-ion spray, but
we didnot have this and, as time goes by, we forgot to do it. We donot
longer analyze MAPS by MS (neitehr ESI, nor MALDI) if we are in doubt we
sequence them. Also in CZE the peaks look very bad, broad with long
tailing.
This is our special experience and as far as I remeber I have not seen any
new results from anyone.
Yours
Manfred
*************************************************
Manfred Raida
Niedersaechsisches Institut fuer Peptid-Forschung
(IPF)
Dep. of Analytical Peptide-Chemistry
Feodor-Lynen Strasse 31
D-30625 Hannover
Germany
E-Mail Manfred_Raida@compuserve.com
Tel.: +49 511 5466 210
Fax: +49 511 5466 102
**************************************************
Ken wrote:
Dear ABRF Colleagues,

I have been asked to analyze (for QC purposes) some MAPS peptides by MS. I
have both MALDI and ESI-triple quad MS available. The expected MW of them
is around 25kDa.

I seem to recall from the dark recesses of my memory that there are some
unique difficulties with this class of peptide. Could anyone with specific
experience with mass spectrometry of MAPS peptides share their experience
with me?

Regards...Ken

********************************

Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA