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Re: [ccp4bb] MS for verification of protein constructs |
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CCP4bb navigationCCP4bb <-- 2007 <-- September 2007 <-- 05 September 2007Subject: Re: MS for verification of protein constructs From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL Date: 2007-09-05 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 > 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 > > pat.loll@drexel.edu > > ===========End of original message text=========== > > > > *********************************** > Jacob Keller > Northwestern University > 6541 N. Francisco #3 > Chicago IL 60645 > (847)491-2438 > j-keller2@northwestern.edu > *********************************** > CCP4bb navigationCCP4bb <-- 2007 <-- September 2007 <-- 05 September 2007 |
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