H/D Exchange Kinetics: Experimental Evidence for Formation of Different b Fragment Ion Conformers/Isomers During the Gas-Phase Peptide Sequencing

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Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) combined with H/D exchange reactions was utilized to explore the existence of different b(5)(+) and b(4)(+) fragment ion conformers/isomers of hexapeptide WHWLQL in the gas phase. Distinct H/D exchange trends for protonated WHWLQL ([M + H](+)) and its b(5)(+) and b(4)(+) fragment ions (with ND(3)) were observed. Isolated (12)C(all) isotopomers of both b(5)(+) and b(4)(+) fragment ions yielded bimodal distributions of H/D exchanged product ions. The H/D exchange reaction kinetics also confirmed that b(5)(+) and b(4)(+) fragment ions exist as combination of slow-exchanging ("s") and fast-exchanging ("f") species. The calculated rate constant for the first labile hydrogen exchange of [M + H](+) (k([M + H](+)) = 3.80 +/- 0.7 x 10(-10) cm(3) mol(-1) s(-1)) was approximately 30 and approximately 5 times greater than those for the "s" and "f" species of b(5)(+), respectively. Data from H/D exchange of isolated "s" species at longer ND(3) reaction times confirmed the existence of different conformers or isomers for b(5)(+) fragment ions. The sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) of WHWLQL combined with the H/D exchange reactions indicate that "s" and "f" species of b(5)(+) and b(4)(+) fragment ions can be produced in the ICR cell as well as the ESI source. The significance of these observations for detailed understanding of protein sequencing and ion fragmentation pathways is discussed.