Hiya
For verification purposes we almost always N-terminal sequence (we are fortunate in that we have such a facility on site so that the samples can be turned round fast) - old technology but good and solid! We then usually combine these data with a Mass Spectra (MALDI-ToF-Tof - on site - is usually accurate enough to see 1-2 amino acid difference) to check for C-terminal trimming, PTMs / other issues. We usually perform these experiments both on purified material and crystals run on a gel, particularly if Captain Paranoid has paid me a visit and we're always if we are about to embark or MIR or MAD etc....(someone once told me a horror story of battling for years to purify a protein and solve a structure only to find it was a lysozome contaminant - probably an urban myth but scary enough)_
J
Anastassis Perrakis <[log in to unmask]> wrote:>
> ... all these are correct indeed - but ESI-TOF is also a nice
> solution, especially coupled to an LC system.
> My understanding was that MALDI-TOF is better for smaller fragments,
> accuracy can be about 10 Dalton ...
>
> for more info there is a useful short review of the use of ms
> techniques for structural work in:
>
> http://journals.iucr.org/d/issues/2006/10/00/gx5084/index.html
>
> (the 'spine' issue).
>
> A.
>
> On 5 Sep 2007, at 22:56, Joel Guenther wrote:
>
>> If you have a very pure protein sample, you'll want to use an ESI-
>> ion trap for analyzing proteins of that size. It should be
>> possible to get an exact mass (i.e. within a single Da). It's
>> possible, but very rare, to get exact masses of proteins up to 100
>> kDa using ESI-ion trap instruments.
>>
>> If your sample is not highly purified, you'll need to use some type
>> of TOF instrument. MALDI-TOF should work. With a TOF instrument,
>> you shouldn't expect to be able to distinguish between point
>> mutants of a protein, but you'll be able to get information about
>> larger changes. For instance, you'll be able to determine if you
>> completely cleaved a his-tag off a construct.
>>
>> Our lab is spoiled because we have access to the HHMI Mass Spec
>> facility, but I'd imagine that there are many facilities that can
>> get accurate masses on 40 kDa proteins.
>>
>> -Joel
>>
>> =================================================
>> Joel M. Guenther
>> PhD Candidate, Department of Chemistry
>> Kuriyan Laboratory
>> http://jkweb.berkeley.edu/
>> University of California, Berkeley
>> 176 Stanley Hall, QB3
>> Berkeley, CA 94720-3220
>> tel: (510) 643 0166
>> fax: (510) 643 2352
>> =================================================
>>
>>
>>
>> On 9/5/07, Jacob Keller <[log in to unmask] > wrote:
>> I second Dr. Loll's question, and would like to be CC'd in whatever
>> MS tips, including
>> service-providers, are sent. I have been having a bit of a debacle
>> with a certain MS service provider.
>>
>> Jacob Keller
>>
>> ==============Original message text===============
>> On Wed, 05 Sep 2007 11:41:52 am CDT Patrick Loll wrote:
>>
>> I wonder if anyone would care to share experiences/ideas/biases that
>> relate to the use of mass spectrometry to verify the identity of
>> protein constructs used for crystallization. Our experience with
>> different MS facilities has been checquered.
>>
>> Specifically:
>>
>> What's the current thinking on the best approach to get
>> masses for
>> intact proteins of moderate size (say, 40 kD)? ESI-TOF?
>> What kind of resolution should one hope to obtain in such
>> cases
>> (10E-04?)
>>
>> Any suggestions as to good facilities offering fee for service MS
>> characterization are welcome (but should be shared off line, I think;
>> continental US only).
>>
>> Thanks,
>>
>> Pat
>> ----------------------------------------------------------------------
>> --
>> ---------------
>> Patrick J. Loll, Ph. D.
>> (215) 762-7706
>> Associate Professor FAX: (215)
>> 762-4452
>> Department of Biochemistry & Molecular Biology
>> Director, Biochemistry Graduate Program
>> Drexel University College of Medicine
>> Room 10-102 New College Building
>> 245 N. 15th St., Mailstop 497
>> Philadelphia, PA 19102-1192 USA
>>
>> [log in to unmask]
>>
>> ===========End of original message text===========
>>
>>
>>
>> ***********************************
>> Jacob Keller
>> Northwestern University
>> 6541 N. Francisco #3
>> Chicago IL 60645
>> (847)491-2438
>> [log in to unmask]
>> ***********************************
>>
--
Professor James Whisstock
NHMRC Principal Research Fellow / Monash University Senior Logan fellow
Department of Biochemistry and Molecular Biology
Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia
+613 9905 3747 (Phone)
+613 9905 4699 (Fax)
+61 418 170 585 (Mobile)
|