PRIMARY STRUCTURES OF PROTEINS CHARACTERIZED BY PROTEINASE-K DIGESTION AND MATRIX-ASSISTED-LASER-DESORPTION IONIZATION MASS-SPECTROMETRY/

To improve the sequence ions of a protein in matrix-assisted laser des orption/ionization mass spectrometry (MALDI-MS), proteinase K was used to digest the protein followed by MALDI-MS characterization of the pe ptide fragments. The primary structures of three proteins, insulin B c hain, cytochrome c and lysozyme, were determined by this method. A ser ies of peptide fragments including those differentiated by one residue can be produced from the protein by using proteinase K digestion, thu s providing support to the protein sequence, The peptide fragments lib erated from proteinase K proteolysis of the insulin B chain allow the protein to be partially sequenced. Furthermore, some of the residues a re double or triple checked by generating a variety of fragments, The same method was used to investigate cytochrome c and lysozyme denatura ted in 3 M guanidine hydrochloride. The success of the method relies o n the intrinsic properties of proteinase K and accurate determination of the peptide fragments by MALDI-MS.