Kate,
We use the following for most of our in-gel digests. It is a slight
modification of the following ref and uses zip tips for sample
clean-up/desalting:
Arnott, D.; OíConnell, K. L.; King, K. L.; Stultz, J. T. Anal. Biochem.
(1998) 258, 1-18.
Additional Refs that may be helpful include:
R. Ogorzalek Loo et al, Electrophoresis 1997, 18, 382-390.
R. Ogorzalek Loo et al, Anal. Chem. 1996, 68, 1910-1917.
A. Shevchenko et al, Anal. Chem. 1996, 68, 850-858.
E. Mortz et al, Biological Mass Spectrom., 1994, 23(5), 249-261.
J. Rosenfeld et al, Anal. Biochem. (1992), 203, 173-179.
Special notes on ASP-N. We used sequence grade Boehringer Mannheim ASP-N
that had been resuspended per their directions. We also noticed
significant cleavage at glutamic acid as well as apartic acid; which is not
uncommon for this protease.
Hope this is helpful.
In-Gel Digestion Procedure
1. Excise native, SDS, satined, or unstained gel bands.
2. Wash twice with 50% CH3CN:50%100 mM NH4HCO3
3. Reduce/alkylate with 25ul of 10mM DTT in 100mM NH4HCO3 for 1hr at 50C
4. Add an equal volume of 50mM iodoacetamide in 100mM NH4HCO3 for 45 min
in the dark at room temp.
4. Remove excess buffer and wash twice with 10ul of 50% v/v MeCN/100mM
NH4HCO3
6. Partially dry gel pieces by vacuum centrifugation
7. Reswell gel with 0.5 ug trypsin in 5-10 uL 50 mM NH4HCO3 for 10 minutes.
8. Add 50 mL 50 mM NH4HCO3 and digest overnight @ 37oC for trypsin and
Asp-n, ambient for chymotrypsin.
9. Retain supernatant and extract gel with 50mL each (a) 50% CH3CN in 1%
formic acid and (b)100% CH3CN.
10. Reduce volume to 5-10 mL by vacuum centrifugation.
11. Pre-Wash Zip tip with acetonitrile and 0.1% TFA.
12. Add equal volume of 0.1% TFA to the analyte and load Millipore C18 Zip
Tip by repetitive flushing.
13. Wash bound analyte with 0.1% TFA.
14. Elute peptides from Zip Tip with matrix solution (24mg/mL in 80%:20%
methanol:water) or sequential fractionation using increasing percentages of
MeCN in 0.1%TFA.
Best Regards,
Lloyd W. Sumner, Ph.D.
Associate Director, The Laboratory for Biological Mass Spectrometry
Texas A&M University
Department of Chemistry
P.O. Box 30012
College Station, TX 77842-3012
Voice #409-845-8404
Fax #409-845-4719
Email Sumner@mail.chem.tamu.edu
For more information concerning the LBMS
and the Applied MS Lab visit our webpages at:
http://www.chem.tamu.edu/rgroup/russell/
http://www.chem.tamu.edu/services/ms/
For more information on The Center for Chemical
Characterization and Analysis see:
http://www.chem.tamu.edu/CCCA/