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Free keywords:
MULTIENZYME DIGESTION FASP; MASS-SPECTROMETRY; TOTAL PROTEIN; DEPTH;
REVEALS; NUMBERBiochemistry & Molecular Biology;
Abstract:
Filter-aided sample preparation (FASP) is a versatile and efficient way of processing protein extracts for bottom-up proteomic analysis. The method repurposes centrifugal ultrafiltration concentrators for removal of detergents, protein cleavage, and isolation of pure peptide fractions. FASP can be used for protein cleavage with different proteinases either with single enzymes or in a mode of successive multienzyme digestion (MED)-FASP. The FASP methods are useful for processing of samples ranging in their sizes from submicrogram to several milligram amounts of total protein. They also allow peptide fractionation, and isolation and quantitation of total RNA and DNA acid contents. This chapter describes principles, limitations, and applications of FASP. Additionally detailed FASP and MED-FASP protocols are provided.
