Re: silver destaining and ES

rjohnson@immunex.com
Fri, 20 Aug 1999 09:51:14 -0700

Roland:
I tried the silver destaining on 100 ng bands of BSA and bovine carbonic
anhydrase, and, compared to not destaining, I found very little difference in
the Maldi spectra. I also spiked the digests w/ a bit of glu fib peptide as an
internal standard, and by LC/MS it seemed that the yields for both BSA and CA
went up by about 50%. Given that I can get different Maldi ion intensities from
the same sample, but spotted twice, I tend to believe the LC/MS results; namely,
that there is a moderate improvement in yield. Since the procedure is fairly
simple, I am leaning towards using the destaining more frequently.
However, I did make a strange observation that I cannot explain. The 100
ng band of BSA turned clear while "destaining", but 50, 25, and 12.5 ng bands
turned dark blue (they were originally orange-brown when stained). I rummaged
through the trash to retrieve additional 100 ng BSA bands that I threw out (I
explained to startled lab mates that I was looking for research ideas), and
these also turned clear upon destaining. The 100 ng band of CA was originally
greyish, but turned dark blue when the destain was added. I'm still puzzling
over that, and I've not repeated it. If anyone could explain this to me, I'd be
interested.

Rich Johnson (Immunex, Seattle)

Roland_S_Annan@SBPHRD.com on 08/20/99 06:40:25 AM



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Subject: silver destaining and ES

The few recent comments on the destaining of sliver stained bands only
discussed using MALDI for the analysis of the peptides. Has anyone tried
using nanoES or LC-MS/MS after destaining the silver stained spots. I'm
not sure why there would be any difference, but I was just wondering. For
those bands where we only get 1-2 peptide sequences, I'd be happy if the
destaining could get us a few more.

Roland S. Annan
Biological Mass Spectrometry
SmithKline Beecham Pharm