Mass Spectrometry of Peptides and Proteins: Fragmentation Pathways of Protonated Peptides Containing Histidine and Conformational Dynamics of Proteins

Theoretical calculations at the B3-LYP/6-31G(d)//B3-LYP/6-31G(d) level on protonated HG and GH model systems indicate that formation of b2(oxazolone-His) ions with the classical oxazolone structure is the most energetically and entropically (kinetically) favored b2 ion formation pathway, irrespective of the location of the histidine. Even though the b 2(diketo-His) ions with the protonated diketopiperazine structure is the most stable, it is the least energetically and kinetically favored pathway. Formation of the b2(bicyclic-His) ions from protonated GH with histidine at the C-terminus is competitive with b2(oxazolone-His) ion formation but shows a higher energy barrier. The theoretical findings are consistent with observed fragmentation behavior of the b 2 ions in energy-resolved tandem mass spectrometric (MS/MS) studies on HGG/HAOMe/HFOMe and GHG/AHOMe/FHOMe.;The initially formed b2(oxazolone-His) ion could isomerize (or cyclize) to b2(diketo-His) ions if additional internal energy is imparted via collisional activation. On the other hand, the b2(diketo-His) ions could be converted back to b2(oxazolone-His) ions by cleavage of the ring amide bonds, ring opening and intra-molecular rearrangements. At relatively long ion trapping times (up to 200 milliseconds), isomerization tends to reach a steady state in which the relative populations of the bi(oxazolone) ions and bi(diketo) ions resembles that of protonated cyclo-(GH) having the cyclic diketopiperazine structure. This is one of the major findings in the present study because the interconversion between bi(oxazolone) and bi(diketo) ions (i = 2 and 3) has not been found for most peptides reported in the literature.;Aside from the b2 ions, unique formation of a non-sequence ion, bn(dehydration, -H2O) ion (for n = 2 - 5), resulting from elimination of a water molecule involving a peptide backbone (amide) oxygen, was also commonly found in the MS/MS spectra of protonated histidine-containing peptides. Based on the dissociation pathways of protonated GH and HG probed by M.O. calculations, we found that the formation of b 2(dehydration, -H2O) ions are catalyzed by the basic imidazole in the side chain of histidine. Furthermore, the ion trap MS3 studies on the b3(dehydration, -H2O) ions of protonated HGG, GHG and GGH showed the loss of different small neutrals (mostly H 2O, NH3 and CO). These experimental and theoretical results demonstrate that the formation pathways, ion structures, and dissociations of bn(dehydration, -H2O) ions vary with histidine at different positions of the peptide.;We have extended the practical application of tandem mass spectrometry to sequencing of proteolytic peptides of a protein-based biosensor, TEM-52f. The TEM-52f biosensor was developed to detect trace levels of beta-lactam antibiotics in contaminated food and diary products. The conformation changes and biosensing mechanism of TEM-52f upon binding to a beta-lactam antibiotic, penicillin G (pen G), was probed by electrospray ionization mass spectrometry and hydrogen/deuterium exchange kinetics. The TEM-52f biosensor was prepared by replacing the valine at position 216 of a class A beta-lactamase mutant (TEM-52) with a cysteine residue, and then a fluorophore, fluorescein-5-maleimide (fluorescein), was attached to this cysteine. The site of fluorescein attachment was determined by proteolytic digestion of TEM-52f, followed by tandem mass spectrometric (MS/MS) and sequence analysis of the individual peptides generated. (Abstract shortened by UMI.).