The sensitivity on a MS can not be "set up", but the difference between
detecting or not detecting an analyte will be dependent on quite a lot of
factors, the three most important ones being sample preparation, column i.d. and
solvent system.
>
>
> I would like to cut bands out of an SDS gels and run them on a HPLC
> attached to a ESIMS. If your facility does this, I would like to know how
> sensitivity your set up is. Specifically, if I have recovered 5 pmols of a
> protein or protein digest from the gel, can you see these
> peptides/proteins in the MS given your current HPLC/MS configuration?
> Can you see 1 , 0.5 or 0.2 pmol? How low can you go before your
> signal-to-noise ratio drops below 10 (or what ever ratio you feel
> confident will provide reliable MW determinations)?
>
>
Starting from your experiment, you said that you recovered approx. 5 pmol of
digested material (you did not say in how much volume, though). For this amount,
I would recomend the use of a 0.5 mm i.d. column, but in any case not bigger
than 1 mm i.d.. The 0.5 mm i.d. will give you a limit of sensitivity of around
250-500 fmol of peptides by UV at 214 nm and probably around 100 fmol in the MS.
Multiply these numbers by 4 for 1 mm i.d. columns.
The solvent system is quite important for the peptide detection in the MS. Using
0.1% TFA will kill your signal. Use 0.025% TFA if you can, better would be
0.005% HFBA (heptafluorobutyric acid) with 0.1% acetic acid.
Depending on the sample volume you have and how clean it is, you can either load
directly on the colunn or use one of these guard or micro columns to
pre-concentrate your sample. If you think to have non-ionic detergent or SDS in
your sample, use one of the pre-column manufactered by Michrom BioResources for
trapping these nasty components. SDS and non-ionic detergent will kill your
separation and your sensitivity.
A last word on the MS: make sure to optimize your ESI source for the conditions
you are going to use (flow rate, acetonitrile-containing solvents). This will
tremendously help to keep a steady signal during the gradient. ALso, if you are
using a quadrupole instrument, consider to relax the resolution to about 500
over the mass range. This will give you an additional boost in sensitivity
without loosing too much in mass accuracy.
Finally, last but not the least: first try you whole procedure with some kind of
standard to make sure your procedure works and that you have some margin in the
sensitivity you wish to obtain. At the most, your real-world sample is probably
going to look only as good as your worse standard.
If you are interested, I can send you a Mat and Meth. section of a paper due for
publication in one month or so. Otherwise, I wish you good luck.
Axel
-- --------------------------------------------------------------------------------
Axel Ducret, Ph.D. Senior Research Biologist Merck-Frosst Canada Inc. Dept. Biochemistry and Molecular Biology P.O. Box 1005 Pointe-Claire-Dorval PQ H9R 4P8 Canada
tel. + (514) 428-3428 fax + (514) 428-4900