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 <[EMAIL PROTECTED]> 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 <[EMAIL PROTECTED] > 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 >> >> [EMAIL PROTECTED] >> >> ===========End of original message text=========== >> >> >> >> *********************************** >> Jacob Keller >> Northwestern University >> 6541 N. Francisco #3 >> Chicago IL 60645 >> (847)491-2438 >> [EMAIL PROTECTED] >> *********************************** >> -- 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)
