Figure 1.

Enhancements to the FASP workflow. Samples are prepared in 4% SDS and diluted with 8 M urea to dissociate SDS from proteins. Microcon filter units and collection tubes are passivated by overnight incubation with 5% TWEEN-20, followed by thorough washing in MS-grade water. Diluted samples are then applied to passivated Microcon filters for buffer exchanges, eliminating SDS and contaminants. Proteins are alkylated with urea present, followed by successive buffer exchanges. Proteins are digested in the presence of surfactants, with the product peptides liberated by centrifugation. Extracting surfactant into an organic layer leaves behind pure peptides for fractionation or directly analysis by LC–MS.