Direct analysis of the products of sequential cleavages of peptides and proteins affinity-bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry

Consecutive enzymatic reactions on analytes affinity-bound to immobilized m etal ion beads with subsequent direct analysis of the products by matrix-as sisted laser desorption/ionization time-of-flight mass spectrometry have be en used for detecting protein synthesis errors occuring at the N-terminus. The usefulness of this method was demonstrated by analyzing two commerciall y available recombinant HIV proteins with affinity tags at the N-terminus, and histatin-5, a peptide with multiple histidine residues. The high specif icity, sensitivity, and speed of analysis make this method especially usefu l in obtaining N-terminal sequencing information of histidine-tagged recomb inant proteins. (C) 1999 Academic Press.