Wenhe Potentially, mass spectrometry in combination with electron capture dissociation and/or collision-induced dissociation might be helpful. See doi: 10.1007/s13361-013-0662-5.
Best wishes, Klaus ======================================================================= Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham E: k.futte...@bham.ac.uk Edgbaston T: @KFbrumbio Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab ======================================================================= > On 21 Aug 2017, at 04:41, <wenhezhong.xmu....@gmail.com> > <wenhezhong.xmu....@gmail.com> wrote: > > Dear CCP4BB members, > > We would like to identify a ligand that is present in crystal structure > (according to strong positive densities at active site) but absent in > crystallization condition. We already have some candidates in mind based on > our knowledges on this protein but we need to validate further. The general > method we are using now is to use methanal to precipitate protein and extract > ligand from the precipitated protein. Then we analyse the methanol extraction > sample on LC-MS. One problem of this method is that the methanol extraction > will not be 100% efficient which means there is only a small portion of > bound-ligand can be extracted from the protein— particularly if the ligand > binds very tightly to the protein. So I would like to know whether anyone has > experience to efficiently extract tighly-bound ligands from protein for > downstream analysis. One method is to digest protein with protease such as > trypsin. Or use urea to denature the protein. However, these methods require > relatively long processing time which is not optimal when the ligand that we > want to analyse is unstable (degrade overtime). Anyone has more suggestions? > > Thank you! > > Kind regards, > Wenhe
