The long-term goal of the research proposed here is to improve the throughput and accuracy of automated tandem mass spectrometric peptide and protein sequencing. There are three specific aims in the proposal: (1) Continue investigations of enhanced cleavage at acidic amino acidic residues in order to confirm structural features that lead to enhanced cleavage. Use ion mobility (ion chromatography) spectrometry and molecular modeling to confirm whether proposed secondary structure leads to unusual fragmentation. (2) Use the SEQUEST program and known proteins to search for structural features that cause unusual fragmentation patterns. SEQUEST is a program that is generally used to correlate uninterpreted product ion MS/MS spectra with sequences from protein and nucleotide databases. In our study, the SEQUEST program will be used as a tool to identify sequence stretches that give spectra that are not correctly predicted based on the current state of knowledge of peptide dissociation. Perform detailed mechanistic studies to clarify/identify the structural features leading to the unusual fragmentation patterns and develop """"""""rules"""""""" that can be made available for incorporation into automated sequencing protocols. (3) Compare the gas-phase collision- induced dissociation and surface-induced dissociation activation methods to determine whether surface-induced dissociation provides a quantifiable improvement in sequence determination by tandem mass spectrometry. Determine whether sequence identification can be obtained by surface-induced dissociation of small proteins without prior digestion, using chemokines as representative structures. These different, but related, projects are expected to increase our fundamental knowledge of peptide dissociation in the gas phase, improve practical automated sequencing of peptides, and characterize whether the surface-induced dissociation activation method should be pursued as a versatile method for practical sequencing of peptides and proteins.
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