Sample preparation and digestion for proteomic analyses using spin filters.
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 > or = 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.
Manza
Linda L
LL
Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, USA.
Stamer
Sheryl L
SL
Ham
Amy-Joan L
AJ
Codreanu
Simona G
SG
Liebler
Daniel C
DC
D001639:Bicarbonates; D002498:Centrifugation / Q000295:instrumentation / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000302:isolation & purification* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D014357:Trypsin
D001639:Bicarbonates; D010455:Peptides; D011506:Proteins; C027043:ammonium bicarbonate; D014357:Trypsin