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: (bigger) fragment identification of limited proteolysis w/ mass-spec
From: Yong Tang liutang19 {- at -} GMAIL {- dot -} COM
Date: 2007-03-31

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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

Subject: Re: (bigger) fragment identification of limited proteolysis w/ mass-spec
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2007-03-31

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

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CCP4bb <-- 2007 <-- March 2007 <-- 31 March 2007

Previous message:
Subject: (bigger) fragment identification of limited proteolysis w/ mass-spec
From: Yong Tang liutang19 {- at -} GMAIL {- dot -} COM
Date: 2007-03-31

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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