Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/5419
Title: Mass spectrometry of peptides and proteins : fragmentation pathways of protonated peptides containing histidine and conformational dynamics of proteins
Authors: Lau, Pui Yee
Keywords: Mass spectrometry.
Peptides -- Analysis.
Proteins -- Analysis.
Hong Kong Polytechnic University -- Dissertations
Issue Date: 2011
Publisher: The Hong Kong Polytechnic University
Abstract: The b₂ ions formed from protonated peptides containing histidine could have three possible structures, namely the b₂(oxazolone-His) ion with a 5-membered oxazolone ring, the b₂(diketopiperazine-His) ion with a cyclic 6-membered ring dipeptide structure, and the b₂(bicyclic-His) ions (for b₂ ions with histidine located at the C-terminus only, and with an oxazolone ring fused onto the imidazole ring in the side chain of histidine). 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 b₂(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 b₂ 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 b₂(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 H₂O, 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 β-lactam antibiotics in contaminated food and diary products. The conformation changes and biosensing mechanism of TEM-52f upon binding to a β-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 β-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. The TEM-52f biosensor is a more sensitive biosensor than a previously developed β-lactamase based biosensor, PenPC E166Cf, because it yields more intense fluorescence emissions (about 4.7 fold increase versus 2.0 fold increase in PenPC E166Cf) upon binding to pen G. TEM-52f has a larger (wider) active binding domain (pocket) than PenPC E166Cf. We found the H/D exchanged levels of the peptide segments of the binding domain of TEM52f is reduced when compared to that of TEM-52, indicating that the fluorescein label is deeply embedded in the hydrophobic environment inside the active binding domain of TEM-52, thus lowering the H/D exchange capabilities of the peptide segments of the binding domain. Upon binding to pen G, the fluorescein is displaced and become exposed to a much more polar aqueous environment outside the binding domain. It is this ‘spatial displacement’ mechanism and associated drastic changes in environmental polarity (from hydrophobic to a much more hydrophilic environment) surrounding the fluorphore that led to enhanced fluorescence emissions of TEM-52f upon binding to pen G. The H/D exchange results are corroborated by molecular modeling of pen G binding to TEM-52f. The solvent accessible area (SAA), a measure of exposure to polar water solvent molecules, of the fluorescein label was found to increase from 211 Ų to 403 Ų upon binding to pen G. The results of this study also reveal that a crucial criterion for designing a more sensitive β-lactamase based biosensor is to construct a wider active binding site of a β-lactamase based protein.
Description: xxvii, 319 p. : ill. (some col.) ; 30 cm.
PolyU Library Call No.: [THS] LG51 .H577P ABCT 2011 Lau
URI: http://hdl.handle.net/10397/5419
Rights: All rights reserved.
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