ENZYMATIC AND CHEMICAL DIGESTION OF PROTEINS FOR MASS-SPECTROMETRY

This chapter discusses enzymatic and chemical digestion of proteins for mass spectrometry. The standard approach for obtaining partial or complete structures of proteins without resorting to cloning cDNAs is to digest the purified protein with one or two selected endoproteases or by chemical cleavage methods, then to fractionate the resulting peptides by reversed-phase high-performance liquid chromatography (HPLC), and to sequence the resulting peptides. In the past, peptides have been primarily sequenced by automated Edman chemistry, which currently has a sensitivity in the mid to low picomole range. Wherever possible, structures were confirmed by amino acid compositional analysis which revealed the amount of sample present and the relative molar ratios of each amino acid. With the advent of mass spectrometric techniques such as fast atom bombardment mass spectrometry (FAB-MS), it has been possible to obtain molecular weights for the majority of peptides analyzed, even at the low picomole range. With these objectives in mind, the chapter presents methods for obtaining peptide maps at the 100 pmol level on widely available standard proteins (horse cytochrome c and myoglobin). © 1990, Elsevier Inc. All rights reserved.