We use a Micromass Q-TOF instrument as a tool for protein
identification, MSMS sequencing, and glycosylation studies (well,
some phosphorylation, too). For identification stuff I use nanoflow
LC/MS/MS (LC packings ultimate LC) at 200nl/min, For ionization I use
the Newobjective pulled silica needles with distal gold coating. With
this setup I get several good MSMS spectra per protein digest (i.e
per run) at a 50 fmol level. I consider this a quite good
sensitivity.
The spectra are of good quality, and all double charged fragments can
be easily ruled out due to high resolution, which makes the
sequencing much more easier. However, resolution 5000 is plenty for
that. Where I would still like to have even better resolution is when
analyzing a glycopeptide having a mass of 6000, and carrying 5
charges (i.e to get an accurate monoisotopic mass). Also I've got the
impression that the Q-TOF 2 is somwhat more sensitive than our "old"
version. More sensitivity is always nice for protein identification
from silver stained 2D gels.
On the other hand, sometimes it would have been nice to do multiple
MS for glycopeptides, as has been shown to be advantageous by Bruce
and Vernon Reinhold. This would of course require an ion-trap
instrument.
To be onest, if I should pick up an instrument for MSMS sequencing it
would defenetely be a Q-TOF (well, why not a Q-Star, but I am not
too convinced about the SCIEX software...).
Hope this is of any help
-Juhani
> Priority: Urgent
> From: Jim Bloom <Jim.Bloom.B@bayer.com>
> Subject: MS
> Date: Thu, 11 Nov 1999 10:35:23 -0500
> To: Recipients of ABRF List <abrf@aecom.yu.edu>
> When we received our sparkling new single quad ESI-MS seven years ago
> we were overwhelmed...wow could we do stuff we had never dreamed was possible!
> After about two years of experience we became jaded...just think what we could
> do with more sensitivity, accuracy, resolution and if only we could sequence
> those peptides in the map on-line!! So, we are now in the position that we
> will be able to upgrade sometime in the forseeable future and I notice that the
> times have changed. In the "old days" we would purchase a triple quad. Triple
> quads now seem to be passe' and the ion trap has become the work horse. The
> new kid on the block seems to be the orthogonal ESI/TOF. So what to do...what
> to do? I have talked to a trusted vendor of ion traps and was told that if we
> have the money we should buy an othogonal ESI/TOF. We have arrived at the
> subject of this email. Two vendors sell ESI/TOFs: Micromass with their QTOF
> and QTOF2 and PE SCIEX and their QSTAR. I am convinced that both companies
> sell instruments that we would be happy with...however there are always pros
> and cons and I would appreciate any input regarding the following questions:
> 1.) Most of the hype revolves around differences in resolution, 5,000
> vs 7500 vs 10,000 etc. In practical terms, is the difference between 5,000 and
> 7,500 significant...how about 5,000 vs 10,000 (I see one can pay an extra
> $100,000 for the difference).
> 2.) We are buying an instrument to do sequencing...we have no
> experience doing MS sequencing...I am told the correct software will make or
> break us. I am told that one vendor has had excellent software for years and
> the other has not quite released theirs yet. Should we go for the vendor with
> the software (a bird in the hand is worth two in the bush)?
> 3.) How about the intangibles...service, support, robustness etc....
> Does one vendor stand out in these areas?
> 4.) Perhaps the ion trap vendor was being too modest and we should buy
> an ion trap??
> 5.) Finally, assuming we buy a fancy new machine should we dispose of
> our trusty old single quad? Reading the mail I see that we may want to keep it
> so that we can continue to do the classic "Steve Carr" method of SIM analysis
> to figure out which peak in our peptide map contains glycosylated peptides.
>
> Weather inconsequential: the Bay area got its first significant rain for the
> season a few days ago and I turned off the automatic sprinklers for the back
> yard (another gopher/mole season comes to an end).
>
> Jim Bloom
> Bayer Corp
> Berkeley
> 510-705-7760
> jim.bloom.b@bayer.com
>
>
>
>
>
>
>
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