Full metadata record
DC FieldValueLanguage
dc.contributorDepartment of Applied Biology and Chemical Technologyen_US
dc.creatorLau, Pui Yee-
dc.identifier.urihttps://theses.lib.polyu.edu.hk/handle/200/6565-
dc.languageEnglishen_US
dc.publisherHong Kong Polytechnic University-
dc.rightsAll rights reserveden_US
dc.titleMass spectrometry of peptides and proteins : fragmentation pathways of protonated peptides containing histidine and conformational dynamics of proteinsen_US
dcterms.abstractThe 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.en_US
dcterms.abstractWe 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.en_US
dcterms.extentxxvii, 319 p. : ill. (some col.) ; 30 cm.en_US
dcterms.isPartOfPolyU Electronic Thesesen_US
dcterms.issued2011en_US
dcterms.educationalLevelAll Doctorateen_US
dcterms.educationalLevelPh.D.en_US
dcterms.LCSHMass spectrometry.en_US
dcterms.LCSHPeptides -- Analysis.en_US
dcterms.LCSHProteins -- Analysis.en_US
dcterms.LCSHHong Kong Polytechnic University -- Dissertationsen_US
dcterms.accessRightsopen accessen_US

Files in This Item:
File Description SizeFormat 
b25226460.pdfFor All Users2.59 MBAdobe PDFView/Open


Copyright Undertaking

As a bona fide Library user, I declare that:

  1. I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
  2. I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
  3. I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.

By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.

Show simple item record

Please use this identifier to cite or link to this item: https://theses.lib.polyu.edu.hk/handle/200/6565