There is no question that determination of the primary sequence of peptides and proteins is important to our understanding of living systems. The proposed research will address several different questions involving singly and multiply protonated peptides. All of the related projects are designed to increase the current understanding of the hydrogen bonding interactions and fragmentation patterns of activated protonated peptides. The long range goals of this work are to provide additional "rules" that can be used to enhance automated primary sequencing of peptides and proteins by tandem mass spectrometry and, ultimately, to relate information on gas-phase fragmentation patterns and energetics of dissociation to peptide and protein conformation. In the proposed research, internally cold protonated peptides will be formed by electrospray ionization. Mass-selected ions will be activated by collisions with a surface over a range of collision energies. The experiments will probe, as a function of amino acid sequence, (i) the influence of hydrogen bonding on molecular conformation and stability and (ii) the mobility of protons in molecules with different internal energy contents. The detailed data on energetics of dissociation will be used to determine mechanisms of fragmentation and the sequence, size, and charge state dependence of the peptide fragmentation efficiency curves. The mechanistic implications of these results will then be sought. The experimental work will be supported by quantum chemical calculations, including determinations of bond orders and energy partitioning values (diatomic energy contributions), which will be used to predict fragmentation pathways. Insight into the excitation mechanism of SID and into the fundamental aspects of fragmentation of large molecules will also be gained. If the relative energetics of fragmentation efficiency vary significantly with peptide sequence (see preliminary results), a concrete link between energetics of gas phase fragmentation and sequence-specific molecular stabilization will be established.