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[ccp4bb] Summary Re: (bigger) fragment identification of limited proteolysis w/ mass-spec |
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CCP4bb navigationCCP4bb <-- 2007 <-- April 2007 <-- 01 April 2007Subject: Summary Re: (bigger) fragment identification of limited proteolysis w/ mass-spec From: Yong Tang liutang19 {- at -} GMAIL {- dot -} COM Date: 2007-04-01 the tips to be very helpful. The servers/programs that people mostly come up with are listed below. I used MULDI-TOF and Artem has suggested more accurate measurement. And also many people suggested that I should combine N-terminal sequencing with the mass-spec to have unambiguous identification. I was also suggested to cut out the two bands from the SDS-PAGE for further trypsin digestion and another round of mass-spec to help identify the bands. I'm grateful for those experts that are always "on-call", even during the weekend, at this forum, not only to my questions, but to many others. Please see below for more details of the individual responses – thank you all! Sincerely yours, -yong 1) http://prospector.ucsf.edu/prospector/4.0.8/html/msfit.htm 2) http://expasy.org/tools/peptidecutter/ 3) http://bioinformatics.genomicsolutions.com/PawsDL.html 4) http://www.expasy.ch/tools/findpept.html Subject: (bigger) fragment identification of limited proteolysis w/ mass-spec ------------------------ From: Yong Tang Dear all, just a super dummy question: I treated a protein with trypsin, found the protein being degraded into two well-define fragments, ran a sizing column to find them co-elute, sent the peak fraction for mass-spec, got the two masses. Now here is the question - is there any program readily available for me to roughly identify these two (around 20K) fragments with the full-length sequence and these two masses, and of cause, with the fact that I use trypsin to cut it? I checked a lot of programs listed on Expasy to find them mostly dealing with smaller peptide length. Any information would be highly appreciated. Thanks and have a nice weekend, -yong -------- From: Jan Abendroth Yong, try the protein prospector (http://prospector.ucsf.edu/) and click the MS-Digest button. Then chose a small number of missed digests. -------- From: Mark Brooks try Protein prospector (it is listed at Expasy). The best is MsFit: http://prospector.ucsf.edu/prospector/4.0.8/html/msfit.htm It takes a while to learn how to use it. The protein has to be in one of the databases also, but its very good once you can find it! -------- From: Peter Cherepanov To be really sure, run the digested protein (2-20 ug, the more the better) on an SDS-PAGE, transfer onto PVDF membrane and ask someone to do N-terminal sequencing of both bands (aka Edman degradation). This will identify the N-termini, given more or less exact masses that you already know from MALDI you will be able to map both fragments without doubt. You can check our paper - PubMed #15371438 if you want a simple example. you can also cut the bands from your gel and try complete in-gel trypsin digest/mass spec - you will see which peptides are present in which fragment. These method can fail or produce confusing results, because not every tryptic peptide will be detected by mass spec (inefficient ionisation etc) so you will often not see the terminal peptides; and there will always be conflicting peptides, found in both (due to very high sensitivity of mass spec for some peptides). Anything else will be really a guess work. -------- From: Kornelius Zeth there is. Depending on the prcision of the mass. -------- From: Juan Sanchez-Weatherby (UEA) Have a look at this site: http://expasy.org/tools/peptidecutter/ It will give you lots more than one site but it may give you an indication of around where to look for the site. -------- From: Oganesyan, Vaheh You can input your sequence to Expasy server, choose tripsin as an enzyme and see where the software will predict the cleavage. Compare to your experimental data. On the experimental side the best would be to send the cleaved products to N-terminal sequencing. Usually it can read at least first 4-5 aa. -------- From: ezra peisach Don't know of a program for you to use - but end terminal sequencing will tell you where the fragments start.... (assuming you can separate them on gel native/denaturing - or an ion exchange column)... -------- From: Wendy Gordon Try this one from expasy; I used it for my 36kDa protein... http://us.expasy.org/tools/peptidecutter/ -------- From: Jason Hurlbert You might want to look at the program PAWS. It will do exactly what you want, but it only runs under Windows. I've not tried it under WINE in Linux, but it may work in that situation as well. The link to the program is: http://bioinformatics.genomicsolutions.com/PawsDL.html -------- From: Anthony Addlagatta Note that any program would only predict the cutting site based on the primary sequence. What you are seeing is structure dependent. I doubt if there are programs that are smart enough to do that kind of job. -------- From: Artem Evdokimov There are several MS-specific programs that I know of but they're not in the public domain. I have a couple of crude PERL scripts for this and an Excel application written by a former colleague. These are too crude to send out, but I would be happy to run your sequences and masses through them for you. How accurately were the masses determined (hopefully using ESI LC MS and not MALDI-TOF, since the mass accuracy of the latter is not as good)? Do the two masses add up to the total m.w. of the starting protein? If they don't add up this likely means that either a) there are smaller fragments that you're not detecting or b) your original m.w. is not what you expect. I assume you also ran the original protein MS in the same way as the fragments? Is the original protein homogenous? If it is not this makes life quite difficult. Finally, since the two fragments associate tightly enough to co-elute on sizing this probably means that you've cut into an exposed loop or a linker region between tightly associated domains. Best regards, Artem -------- From: tea.pavkov@uni-graz.at did you think about to do (or send your samples) for the edman degradation? -------- From: Meier Chris (SLH) I would suggest you use the FindPept tool: http://www.expasy.ch/tools/findpept.html Just enter your protein sequence and the Mass-spec results of your fragments: PindPept will then calculate all the possible fragments which correspond to this mass. Simply choose the correct one given the known cleavage behavior of your protease (trypsin). Not the most elegant solution, but it works: I have used it successfully for all my limited proteolysis experiments. -------- From: jjwarren@duke.edu There is a program called PAWS that does exactly what you're talking about, and I think there is a freeware version (unfortunately for PC only, it appears): http://bioinformatics.genomicsolutions.com/paws.html -------- From: Juergen Bosch You should be able to solve your problem with the following link: http://us.expasy.org/tools/findpept.html -------- From: Paul Kraft Certainly you can seperate these fragments by SDS-PAGE, cut the bands out, redo the proteolysis and throw them into the MS again... ---------- Forwarded message ---------- From: jjwarren@DUKE.EDU There is a program called PAWS that does exactly what you're talking about, and I think there is a freeware version (unfortunately for PC only, it appears): http://bioinformatics.genomicsolutions.com/paws.html CCP4bb navigationCCP4bb <-- 2007 <-- April 2007 <-- 01 April 2007 |
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