Ken Mitchelhill,
Ken,
You didn't say whether your 100 rat liver 2D gel spots were silver
stained or coomassie stained. Here's what I'd do.
Do the in-gel digest, and use 10% of the sample and get a MALDI map. If
the data looked good, fine. If the data looked weak but promising, I'd
concentrate the 25-100% of the remaining sample to 0.5 uL with a
microcolumn and re-acquire the data. It's not very tough to get more rat
liver.
We acquire the data using time-lag-focusing (delayed extraction) and use
two internal standards. The searches are done using .005-.01 % error
tolerance. We use no MW limits and no species delimiter. The goal is to
get a single answer, or family of answers (like 50 actins). We are 100%
confident if we get a single answer where the matched peptides cover 20-30%
of the sequence. This is much easier for big proteins.
The way we do the first pass search, we should really get either the right
answer or no answer. If we get no answer, we carefully back off of the
criteria. It's possible the sample is a mixture, and then you will need to
accept fewer matches from the total as a hit (but the coverage should still
be no worse than 20-30%). Even this should probably provide only a single
answer, or one that stands out from among a few (3-4 different proteins).
If you have a good healthy data set and you find it necessary to back off
until you start getting 10-25 different proteins with all the same number
of peptide matches, you probably need to resort to sequencing.
Your chances of finding matches are much poorer in the rat because so
little of the genome is known. But the sad truth is that even very faint
spots turn out to be actin, GST, BSA or heat shock proteins, and these are
easily identified by MALDI mass mapping. This is done in less than an hour
rather than wasting time sequencing junk proteins. We recently worked on
5 "unknown" rat liver proteins from silver stained gels and identified
them all using MALDI mass maps. Three of the five were very common
proteins. For 100 rat liver spots I could only guess that your success
rate might be 25-45%.
In the end it's worth doing the MALDI because it's requires so little
sample and so much less time compared to every other technique. You will
eliminate many of the samples and can then concentrate your energies on the
more promising spots.
Roland Annan
SmithKline Beecham