StvTindall <StvTindall@aol.com> on 11/12/99 07:24:16 AM
To: Jim Bloom/BRKL/PH/US/BAYER@BAYER-US-NOTES
cc:
Subject: Re: MS
Jim,
My guess is that about 90% of the responses to your question will be
"private" responses. It doesn't seem that anyone wants to publicly talk
about equipment in the ABRF forum anymore.
It would be really appreciated by myself and others if you posted a
compendium of the "private" postings. Your question is very relevant. I
haven't been able to get straight answers to similar questions, which makes
me wonder what the vendors are hiding from us.
Thanks,
Steve Tindall
Argo BioAnalytica Inc.
Jim,
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
Dear Jim,
since our latest grant application has been accepted, we are now in the
happy situation to buy a QTOF instrument for our lab. For this reason we
have been in Manchester at Micromass as well as in Langen (FRG) at PE to
evaluate both. I will try to give you a concise overview of what was our
impression.
We looked at the QTOF-1 from MM and at the QStar. MM has now released the
QTof2 which is specified with resolution 10000. We did not evaluate what
the actual resolution of QTof1 was because we intend to purchase the 2, but
the QStar reached approx. 9000 with little optimisation and they claimed
that the have instruments in the field with 12000 resolution. Both are
specified with identical sensitivity, which was proved by people from Astra
(Lund, Sweden) in the lab of P. Roepstorff and M. Mann in Odense, where
they got signals with exactly the same dilution of samples. Peter
Roepstorff told me that they assume the sensitivity is not limited by the
instrument itself but by adsorption to the transfer capillary etc. So, in
short: My opinion is that both instruments (QTof2 and QStar) are
technically almost identical. Oh, both instruments support to my knowledge
meanwhile a precursor ion scan but this in both cases a software option, no
real PIS. This means that it is possible to scan for e.g. PO4 or glycans,
but you will definitely not reach the sensitivity of a triple quad.
Regarding the service I talked to someone at Boehringer Ingelheim (FRG) who
has instruments of both vendors in his group. He said that service of both
is good, no complaints. Gitte Neubauer from the EMBL group told me that PE
service is really good, but you have to keep in mind that the EMBL group
has very close contact to the PE applications lab so they are some kind of
a VIP customer. The have a QTof for apprx. one year as well and they did
not need the service so far (which is at least not a bad sign).
Price difference between QTof2 and QStar is neclectable. Both instruments
have a list price of approx. 470.000$ (at least in Germany). Software
package delivered with the instrument is rudimentary.
The point which brought us to a decision towards Micromass is the software
packages additionally available (ProteinLynx, MaxEnt suites) (apart from a
CE compatible ion source). Its is a pitty, but the software from PE is (in
my opinion) currently unacceptable. PE will switch during next year to a
new NT based software while the recent one is running under MacOS.
Unfortunately, they could not show us a demo of the new software nor could
they tell us what features exactly will be enclosed. On the other hand, if
you buy a QStar now, you buy the software switch as well. No chance of
keeping the Mac software, you must move. So what I can tell you about the
Mac software is following: It is not possible to perform LC-MS runs (not
supported by software as the applications chemist told us), the software
has no charge state decryption algorithm and the Apple script which should
perform a automated database search with five MSMS spectra from a peptide
map crashed two out of three times. So far for PE.
We had exactly the same sample (known spot from silver stained 2D gel) with
us in Manchester. They made a 7 min LC/MS-LC-MS/MS run, aqcuired 48 MS/MS
during this period, directed the spectra in an automated database search
and we could a ten page report about our (correctly) identified protein
after 20 min. The software also allows by MaxEnt the charge state
decryption of higher charged MS/MS spectra, which makes manual
interpretation much easier.
So far for the moment. If you need some more information, pleas feel free
to contact me. I will try to give you an answer if possible.
Dear Jim,
here's my subjective opinion given that I have no experience with the
Q-Star, but I will try to present both sides of the situation below.
Regards,
At 10:35 AM 11/11/99 -0500, you wrote:
> 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).
========================
It is my impression that the Q-Tof2 and the Q-Star have very similar specs.
I don't think you can buy the original Q-Tof (somewhat inferior than the
Q-Star) any more, unless you buy a refurbished model.
> 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)?
==============================
Yes, yes, yes! Micromass software works and works very well. I have been
doing peptide sequencing by MS/MS for many years and have dabbled in
programming as well, and their software is elegant, well written, and, most
importantly, useful. With the Q-Star you will have to suffer through the
transition from Mac to NT and, as those who had bought the PerSeptive
software will tell you, delivery dates offered by PE Biosystems are, shall
we say, very flexible.
> 3.) How about the intangibles...service, support, robustness etc....
>Does one vendor stand out in these areas?
=========================================================
Support is probably similar with both. One thing to keep in mind though,
the Q-Tof has been around for 3-4 years and the hardware and software
glitches, that inevitably show up once the instrument has been in the
field, have been addressed for the msot part. As you know, tolerances on
components are very difficult to gauge ahead of time.
> 4.) Perhaps the ion trap vendor was being too modest and we should buy
>an ion trap??
=====================================
You will not get the same performance with the ion trap. Plus, if you have
the money for a Q-Tof2/Q-Star spend it before the bean counters take it
back. It's easier to get $180K later if you really want 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.
>
=================
You might consider getting a nanoflow source for your quad. I can give you
the name of a small company that will build one for you (one that actually
works!) for a modest price. And no, it's not my company.