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Sample preparation and digestion for proteomic analyses using spin filters
Author(s) -
Manza Linda L.,
Stamer Sheryl L.,
Ham AmyJoan L.,
Codreanu Simona G.,
Liebler Daniel C.
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401063
Subject(s) - chromatography , digestion (alchemy) , chemistry , ammonium bicarbonate , centrifugation , sample preparation , mass spectrometry , trypsin , dialysis tubing , membrane , biochemistry , enzyme , raw material , organic chemistry
We describe the use of commercially available microcentrifugation devices (spin filters) for cleanup and digestion of protein samples for mass spectrometry analyses. The protein sample is added to the upper chamber of a spin filter with a ≥ 3000 molecular weight cutoff membrane and then washed prior to resuspension in ammonium bicarbonate. The protein is then reduced, alkylated, and digested with trypsin in the upper chamber and the peptides are recovered by centrifugation through the membrane. The method provides digestion efficiencies comparable to standard in‐solution digests, avoids lengthy dialysis steps, and allows rapid cleanup of samples containing salts, some detergents, and acidic or basic buffers.