Tryptic digest of the excised gel band, followed by rigorous peptide
matching helped us identify a fragment in a very recent case. The
crystallographic results confirmed MS findings.
Curiously, the ends were 'ragged' so direct ESI-MS was not very useful,
likewise the N-terminal sequencing would not have been a good idea because
of the heterogenous N-terminus.
Artem
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Klaus
Futterer
Sent: Tuesday, July 01, 2008 7:11 AM
To: [log in to unmask]
Subject: [ccp4bb] Sequence of crystallised protein fragment
We have a 150 kDa protein that reproducibly crystallises at one of
the Hampton Screen conditions. However, we know from SDS gel analysis
that the crystals contain only a 45 kDa fragment, that forms through
proteolytic cleavage over time. We would like to determine the
sequence boundaries of the fragment.
We believe a combination of N-terminal sequencing plus MS analysis
might give us the information we need, but I was wondering whether
the ccp4bb community has other suggestions.
Klaus
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Klaus Fütterer, Ph.D.
School of Biosciences P: +44-(0)-121-414 5895
University of Birmingham F: +44-(0)-121-414 5925
Edgbaston E: [log in to unmask]
Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/
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