Filter-aided sample preparation for proteome analysis

Abstract

Quantitative protein extraction and high-yield generation of peptides from biological samples are the prerequisite for successful bottom-up type proteomic analysis. Filter aided sample preparation (FASP) is a method for processing of SDS-solubilized cells in a proteomic reactor format. In FASP, disposable centrifugal ultrafiltration units allow for detergent depletion, protein digestion, and isolation of peptides released by proteases from undigested material. Consecutive protein digestion with two or three proteases enables generation of peptide fractions with minimal overlap and considerably increases the number of identifications and protein sequence coverage. FASP is useful for analysis of samples varying in size from a few micrograms to several milligrams of total protein.