Re: Protein Sequencing and MS
Gary Hathaway (hathaway@cco.caltech.edu)
Fri, 27 Feb 1998 03:15:54 -0700
At 11:35 AM -0700 2/26/98, Jim.Bloom.B@bayer.com wrote:
>I very much enjoyed Joe Fernandez's description of his experience with HP and
>ABI sequencers and it got me thinking. We have had HP and ABI sequencers over
>the last five years and no matter who the vendor, protein sequencers
>require an
>inordinate amount of "care and feeding" and wouldn't it be nice if we could
>spend time on productive things rather than keeping the sequencer happy? (I am
>in another room so ours cannot see what I am typing!) I have heard an
>incredible amount of hype from MS vendors and starry eyed MS "experts"
>over the
>years about how MS can replace good old Edman. So, in case the world has
>passed me by and relying on the real world experiences and hopefully lack of
>axes to grind of our ABRF members let me pose the following questions
>regarding
>Edman vs MS: 1.) lets say I am given a glycosylated protein, in solution or
>on a blot, and need to identify it and any contaminants and determine if there
>are any internal clips...can MS replace Edman? 2.) lets say I am doing
>peptide mapping on a known protein and wish to identify all the peaks (to
>verify the sequence and look for unusual post translational modifications) as
>well as to determine N- or O-linked glycosylation sites and while we are at it
>disulfide bonds...can I use MS alone without supplementing it with Edman and
>get unambiguous results? By the way, when I am asking if MS can replace Edman
>I mean to say: can a reasonably intelligent individual lacking infinite time
>decipher the MS results? I will add that we have been the proud owners of a
>single quad ESI-MS for five years and I know it will not do these
>things...so I
>am also asking if some new fangled MS like these things they call "ion traps"
>will do the trick or MALDI-TOF or ESI-TOF or ...(ain't technology great, the
>latest and greatest is obsolete in less than five years...what happened to
>"triple quads"?)
>
>Weather inconsequential: there is a strange yellow orb in the sky over the
>San Francisco Bay area today, I believe this orb has a name but I have
>forgotten what it is...
>
>Jim,
I was drawn by your question's length, the fact that it seems to call for
an opinion, and its thinly veiled cynicsm (is there any chance we're
related?).
1.) kind of a toss up here, but it seems to me you'll get a lot farther
answering this question with MALDI-TOF vs Edman, but a lot depends. For
example, what mass is the protein, how many clips, and are we talking about
stoichiometric clips or a low level of clipping?
2.) you can get ambiguous results here whether you use Edman or MS. Edman
depends on recovering all of the peptides from HPLC or other purification
technique to at least 80 mole per cent pure. We tried this type of
experiment on an expressed protein of about 50 kDa which ran as a doublet
on SDS gels. With a combination of Edman, MALDI-TOF and an LC/MS run on the
ESI-triple quad. we got 86% coverage, and, while MALDI located some
peptides which were not seen in the electrospray, some didn't show in ESI.
Even with MS on every HPLC peak we couldn't cover every peptide, which left
the question of where the two proteins differed unanswered. We are waiting
for undigested sample to get the mass which together with the sequence and
our coverage should allow us to predict the structures.
Now, since you keep referring to posttranslational modifications and
glycosylations, let me ask you: how does one detect these with Edman
anyway? And it's for sure phosphopeptides are a whole lot easier with MS
than Edman.
I guess in the final analysis,there are no guarantees in science no matter
how sophisticated the hardware. So why not stop looking for them? Do your
best, pay your penny, and take your choice, even if it's a triple quad.
regards
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Gary M. Hathaway, Director
PPMAL - Protein/Peptide Micro Analytical Laboratory
California Institute of Technology
139-74, Division of Biology
Pasadena, CA 91125
http://www.cco.caltech.edu/~ppmal
email Gary: hathaway@caltech.edu
email facility: ppmal@caltech.edu
phone: lab (818) 395-6388 or office (818) 395-2769
FAX (818) 449-3414
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