The LNC-NINDS Protein/Peptide Sequencing Facility has continued to provide NINDS investigators with theoretical and technical expertise for the separation, purification, and amino acid sequencing of peptides and proteins. During the past year, a total of 47 service and cooperative projects were worked on. Highlights of two cooperative projects by the Facility are: (1) Myelin-associated glycoprotein (MAG) has been observed to be cleaved in vivo by an endogenous protease at or near the transmembrane section to yield soluble dMAG. Cleavage occurs at an increased rate in multiple sclerosis patients. The cleavage site of human MAG was determined by a combination of proteolytic digestion, microbore HPLC/MS/MS and database searching. The cleavage site was found to be identical to that previously determined by the Facility for bovine MAG by classical methods involving Edman degradation of the C-terminal proteolytic fragment isolated by anhydrotrypsin affinity chromatography and purified by RP-HPLC. The nature of the cleavage site suggested the presence of a previously unreported human protease in CNS. (2) The filament portion of the bacterial organelle of motility, the flagellum, is composed of molecules of a monomeric motor protein, flagellin. Flagellin from two mutant strains and wild type Salmonella typhimurium bacteria was subjected to proteolytic digestion and the resulting digests analyzed by comparative peptide mapping techniques. Peptide peaks that differed in RP-HPLC retention time, indicative of differences in the primary sequence of the parent protein, were subjected to automated Edman degradation. A valine for alanine substitution found by this technique could account for the observed difference of straight- right-handed or straight-left-handed conformation of the flagellar filament. The sequences found are being used in the design of cDNA probes for cloning experiments. During the last year, the Facility acquired and set up major new equipment, the Finnigan MAT LCQ ion trap mass spectrometer coupled to a Michrom microbore HPLC. Mass spectrometry has become an increasingly important technique for the analysis of peptides and proteins. When operated in LC/MS/MS mode, the LCQ can be programmed as an intelligent, on-line detector for peptide mapping experiments and for the analysis of proteolytic digests for the identification of proteins isolated by 1- and 2-D PAGE or on NC or PVDF blots (a major activity of the Facility) and is thus complementary to our present automated Edman degradation equipment and significantly increases sensitivity to identify those proteins available in minute amounts.
