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Re: [ccp4bb] (bigger) fragment identification of limited proteolysis w/ mass-spec
 

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CCP4bb <-- 2007 <-- March 2007 <-- 31 March 2007
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Subject: Re: (bigger) fragment identification of limited proteolysis w/ mass-spec
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2007-03-31 		Next message:
Subject: Re: (bigger) fragment identification of limited proteolysis w/ mass-spec
From: tea {- dot -} pavkov {- at -} UNI-GRAZ {- dot -} AT tea {- dot -} pavkov {- at -} UNI-GRAZ {- dot -} AT
Date: 2007-03-31


Subject: Re: (bigger) fragment identification of limited proteolysis w/ mass-spec
From: jjwarren {- at -} DUKE {- dot -} EDU jjwarren {- at -} DUKE {- dot -} EDU
Date: 2007-03-31
Yong-

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

Josh

Quoting Artem Evdokimov :

> Dear Yong,
>
> 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
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yong
> Tang
> Sent: Saturday, March 31, 2007 1:06 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] (bigger) fragment identification of limited proteolysis w/
> mass-spec
>
> 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
>



_____________________________________________
- Joshua Warren, PhD (jjwarren@duke.edu) -
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