Silver staining and MALDI MS - can they work together? - silver stained 2DE spots = no MALDI MS results (Apr/21/2005 )
i have a question concerning the removal of silver stain from 2DE spots prior to tryptic digestion and maldi mass spectrometry.
We generate large format 2DE gels in our lab which we stain with the Invitrogen SilverQuest silver staining kit prior to mass spec analysis of our spots. This stain comes with the claim that it is mass spec friendly yet when we excise our spots and send them off to our mass spec lab, they typically come back to us with about a 30% hit rate from our silver stained spots. They employ their own destain procedure (sodium thiosulfate/potassium ferricyanide) prior to tryptic digestion which indeed may be slightly different to the destain reagents in the kit, however the last time we submitted spots for maldi analysis i decided to destain them with the kit reagents, and we ended up with approx 50% of our proteins having some sort of identification. Additionally we had the grumbling of the lab fellows as they said there was stupidly high background which completely removed any hope of an automated database search process because of the generation of spurious peaks. For us though, 50% identification was mildly better than before.
I'd like to think there is a way to enjoy even more success in identifying silver stained proteins by maldi mass spec, but am not sure just what the method might be. To that end i was hoping some wise person amongst you all might be able to part with some advice, either pertaining to the SilverQuest kit specifcally, or to silver stains in general (ie what's the lifetime of silver stained spots before they become too badly modified to beuseful? is it better to destain or not and what is the method? etc etc).
We really want to know what our proteins are, but as you can possibly imagine it is mildly soul destroying when we can routinely cut 150 beautifully resolved spots from our gels, but just as routinely end up with 100 of them generating rubbish peptide spectra.
Any help would be gratefully received.
i have never done mass-spec before, but going to proceed with that. as far as i concern, in order to send the silver-stained spot for mass-spec, the silver staining procedure shouldn't include glutaraldehyde which will affect the mass-spec result. does ur silver staining kit have glutaraldehyde? the silver staining procedure described by Shevchenko, A et al. Anal Chem. 1996 68(5): 850-8 was normally practiced in our lab. silver-stained spot was must be destained before proceding to mass-spec, while coomassie-satined spot doesn't need. the destaining procedure used here is by utilizing sodium thiosulfate/ potassium ferricyanate.
The fixation step in silver staining is the most important step, were extended fixation with Glutarladehyde/formaldehyde will result in permently cross linking th eproteins to the gel and becomes non-recoverable. Personaly, I have used the Bio-RAD Sliver staining kit, which they also claim that it is mass spec comaptible. However, to get it to work for me I had to optimize the conditions for it so I can get low back ground with out reducing the sensitivity, therefore I reduced their recommended fixation step up to half of the recommended time and reduced the amounts recommended for develping one gel to half as well and I got very thus got 75% ms spec recovery with very good signal to noise ratio.
i hope this information is of hel to you, good Luck!