Dear Axel
With reference to Your mail reply to the abrf mailing list (see
below) , I would like to know more precisely the solvent compositions
of HBFA/ HAC system You use ( 0.005% HFBA + 0.1 % HAC in A, but how
about the B solvent?) what is the quality of the UV trace?
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> 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
>
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Thank You
-Juhani
Juhani Saarinen
Protein Chemistry Laboratory
Institute of Biotechnology
University of Helsinki, Finland
e-mail jksaarin@operoni.helsinki.fi
Phone +358-9-708 59 414
Fax +358-9-708 59 416
Street adress:
Viikinkaari 9
FIN-00014, Helsingin yliopisto
PL 56