We propose to establish a facility for micro-sequencing of proteins and peptides utilizing a gas-phase sequencer and a state-of-the-art high performance liquid chromatograph. At least eight investigators will use this facility for a variety of projects: 1) The primary structures and sites of phosphorylation and methylation of nucleolar nonhistone proteins C23, B23 and 160K will be analyzed. The facility will also be used in studying regions of interaction of these proteins with other macromolecules. 2) Peptides from the carboxyl-terminal region of eukaryotic polypeptide chain initiation factors eIF-2(Alpha) and eIF-4A will be sequenced. 3) Sequence information will be obtained on a 78 kDa poly(A) binding protein and the location of methylated lysine residues will be determined on elongation factor Tu from murine erythroleukemia cells. 4) Portions of amino-acyl-tRNA synthetases from the silkworm B. mori will be sequenced and active site regions will be identified after affinity labeling. 5) The facility will be used to assess the structural homogeneity of the Mu and L chains of mouse monoclonal antibodies. 6) To determine whether separate species of enzyme exist for each metal cofactor, sequence data will be obtained on manganese and iron superoxide dismutases from S. mutans. 7) Domains from catfish immunoglobulins will be sequenced to determine the structural basis for the distinct H and L chain subclasses. In projects 1 through 4 obtaining accurate sequence information is essential for cloning and DNA sequencing projects. In all projects the amounts of obtainable protein are limited, thereby creating the necessity for a high sensitivity protein sequencing facility.
