Kevin
You might consider looking at the following abrf archive
posting...
http://www.abrf.org/archives/hmail/0014/0080.html
High salt is an issue for sure. ESI does not like it.
The sample may not be as pure as you hope and contain
multiple species. This leads to spreading the ion current
out over many m/z values. Even if 'pure' the sample may
be micro-heterogeneous (lots of different glycoforms).
It may also contain many different types/amounts of salts
adducted (spreading out the signal). The juicy part of
envelope may be outside the mass range you are scanning.
Some MAbs I have run show most ion-current from 25-4000!
You probably need to run it in profile mode and average
the scans. Profile gives 10-20 data points/amu rather than
the 1/amu that centroided data provides.
I also often ONLY made nice data when running the really
large proteins in LC/MS mode. A VERY large pore (1-4000A)
PLRP (non-silica based RP) run with a short de-salting
gradient using 1% acetic or 0.2% formic acid (not tfa).
You can inject in TFA to keep it in solution...but maybe
not 'intact'. (Thanks to K.Nugent and Mark Hail for this
'trick'). Kerry Nugent's Michrom company sells the PLRP
in a variety of IDs.
As you might infer...the chance of failure is great given
all the possible 'issues'. For a simple ID...try a digest
followed by data base searching...then check the refs in the
url above to try to get the multi-meric solution state.
I have not tried one by nano-spray techniques but that may
also be interesting and fast to attempt-but desalt if poss.
A difficult sample at best.
Matt Sweeney
mattsweeney@earthlink.net
Mass Spec Consulting
Training/Operations/Consulting/Method Development
LC/MS Pharmacokinetics, Peptides, Proteins, Metabolism,
Maintenance Classes, Specialist in Finnigan Equipment and Software
-----Original Message-----
From: Kevin Howland <k.howland@ukc.ac.uk>
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Date: Monday, February 19, 2001 4:02 AM
Subject: MS: High Molecular Weight Proteins
>I'm trying to do some MS on a large (>220 kDa) protein that may or may not
>exist in multimeric form and am not having much joy/success. I get an
>increase in TIC whenever I introduce the sample into the MS but can't seem
>to see a discernable mass/charge envelope. (N.B. It needs high salt
>conditions to keep it in aqueous solution).
>
>Any suggestions or lit references that may help will be gratefully received.
>
>Thanks in advance
>
>Kevin
>
>_______________________________________
>Kevin Howland
>
>Protein Science Facility Manager
>Research School of Biosciences
>University of Kent
>Canterbury CT2 7NJ
>Tel: +44-1227-764000 ext 7987
>Fax: +44-1227-763912
>email: k.howland@ukc.ac.uk
>
>
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