Re: MS/digest

Christian Lombardo (crl@burnham-inst.org)
Tue, 5 Oct 1999 08:47:40 -0700

>Hi, all,
>We are trying to do digests on 1 dimensional gels that were run under the
>following conditions: Laemmli gels were stained with Coomassie Blue (50%
>methanol, 10% acetic acid, 0.2% R-250 Coomassie Blue, and destained
>with 50% ethanol 10% acetic acid and then stored in 10% acetic acid.
>
>Should we be able to get tryptic digestion (using, say Ulf Hellman's
>protocol) and retrieve fragments from proteins bands in this gel--or do
>these stain and destain conditions "fix" the protein in the gel so well
>that we will not be able to get either digestion of the protein
>by the trypsin or extraction of the fragments from the gel pieces.
>
>We actually are trying to do CNBr cleavage of the proteins in-gel--as we
>know the chemical cleavage can occur--but are not having good luck
>finding fragments by MALDI. CNBr fragmentation of protein in solution
>worked well with good MALDI results.
>
>In the coomassie blue stained gel, we are having trouble removing the
>coomassie blue--is that a result of our staining method?
>
>Thanks,
>Deb McMillen
>Institute of MOlecular Biology
>University of Oregon
>Eugene OR

Hi Deb,

My experience with technique to date indicates that the concentrations of
acetic acid can be a factor depending how on how far in sensitivity you
need to go. Higher concentrations of acetic acid will have a greater
tendecy to fix the protein into the gel. We use 0.2% CB G250, 20%
methanol, 0.5% acetic acid for the staining solution. We use 30% methanol
for destaining, no acetic acid. We store the gels in 0.5% acetic acid.
This is similar to the ABRF protocol that is posted with the only
difference being that we use CB G250 vs. R250. All this being said, folks
still bring gels to our Facility for MS identification where they have
performed a CB staining protocol similar to what you are using. In most
cases, we are able to ID the protein by MALDI tryptic peptide mass mapping
if there is enough material there, say at least 5-10 pmol. You will
probably get better results, though, if you reduce the acetic acid
concentrations.

Another point worth mentioning is that the larger fragments produced by
CNBr cleavage are going to be tougher to extract out of the gel. This
could explain your absence of peaks in MALDI. There is a nice paper by
Cohen and Chait, Analytical Biochemistry vol. 247, pp257-267 (1997) that
describes a method of eluting proteins out of gels with a saturated matrix
solution. This might be helpful for your situation.

Regarding complete removal of the CB stain from the gel slice: Several
in-gel digest protocols I have read recommend additional washing steps to
completely remove the CB dye before proceeding with further steps of
reduction/alkylation and tryptic digestion. Hope this helps.

-Chris-

***********************************
Christian R. Lombardo, PhD
Director, Molecular Analysis Facility
The Burnham Institute
10901 North Torrey Pines Road
La Jolla, CA 92037
858-646-3108
crl@burnham-inst.org
***********************************