Re: [ccp4bb] Sequence of crystallised protein fragment
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:[EMAIL PROTECTED] On Behalf Of Klaus Futterer Sent: Tuesday, July 01, 2008 7:11 AM To: CCP4BB@JISCMAIL.AC.UK 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 - Klaus Fütterer, Ph.D. School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: [EMAIL PROTECTED] Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ -
Re: [ccp4bb] Sequence of crystallised protein fragment
ESI-MS at a precision of +-2 Da should work alone. Best wishes Kornelius On Tue, 1 Jul 2008 19:32:56 +0100 Matthew Chu <[EMAIL PROTECTED]> wrote: > N-terminal sequencing / MS for intact mass analysis are the only ways that I > can think of. > > Matt > > 2008/7/1 Klaus Futterer <[EMAIL PROTECTED]>: > > > 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 > > > > > > > > > > > > > > - > > > >Klaus Fütterer, Ph.D. > > > > School of Biosciences P: +44-(0)-121-414 5895 > > University of Birmingham F: +44-(0)-121-414 5925 > > Edgbaston E: [EMAIL PROTECTED] > > Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ > > - > > > > > > > > Matthew LH Chu > PhD Student > School of Pharmacy and Pharmaceutical Sciences > University of Manchester > -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany [EMAIL PROTECTED] Tel -49 7071 601 323 Fax -49 7071 601 349
Re: [ccp4bb] Sequence of crystallised protein fragment
N-terminal sequencing / MS for intact mass analysis are the only ways that I can think of. Matt 2008/7/1 Klaus Futterer <[EMAIL PROTECTED]>: > 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 > > > > > > > - > >Klaus Fütterer, Ph.D. > > School of Biosciences P: +44-(0)-121-414 5895 > University of Birmingham F: +44-(0)-121-414 5925 > Edgbaston E: [EMAIL PROTECTED] > Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ > - > Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
[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 - Klaus Fütterer, Ph.D. School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: [EMAIL PROTECTED] Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ -
