Vijay,
It is hard to guess what you mean when you say that 'mass spec results confirmed both protein bands are the same'. Do you mean that both bands were cut, digested with e.g. trypsin, and the resulting peptides were mapped? Or, alternatively, you've done an MS spectrum on the sample that went on the gel, and found only one species? If it is the first case then the two bands are not necessarily (chemically) 'the same' because peptide mapping is almost never complete, and certain types of PTM cannot be detected like that. Therefore you should perform the other experiment (i.e. direct MS of the sample using a high-resolution ESI-MS instrument) and see what protein species are present in the sample. If it is the second case, i.e. you've already done the direct mass determination and found no obvious signs that there are two species present, then the situation is slightly more challenging. The interpretation of MS results depends on the quality of the spectrum both in terms of signal/noise ratio and in terms of the resolution of the instrument. It also depends on the size of the protein - typically larger proteins are more difficult and additional chemical species can slip by undetected. If your experiment results are confident to within a few daltons, then you probably have a 'subtle' event such as deamidation of an amide, resulting in an acid side chain (and an additional charge), a redox state change (S-S bond is only 2 Da different from SH SH pair), or a conformational change that persists even in SDS-PAGE (some more stubborn proteins can remain fully or partially folded even in SDS). Also, you can have a strong metal binding which would persist through SDS-PAGE but may be dislodged by TFA, sinapinic acid, etc. during the MS experiment. There are other unstable modifications that may be noticeable on the gel but completely (and artefactually) destroyed by the MS. Likewise, there are gel artefacts e.g. associated with DP clipping at higher temperatures, that may not appear on MS. If you only did e.g. MALDI-TOF kind of experiment, then your resolution may be low enough (especially for larger proteins) that changes in one to a few amino acids may slip by undetected. Clearly, the solution here is to do the other kind of MS, to see if at higher resolution things don't look more interesting. Hope this helps, Artem _____ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Vijay Kumar Sent: Sunday, November 04, 2007 7:22 AM To: [email protected] Subject: [ccp4bb] protein degradation? Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay
