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postgraduate thesis: Multidimensional liquid chromatography/mass spectrometric analysis of selected post-translationally modified peptides : from fundamentals to shotgun proteomics

TitleMultidimensional liquid chromatography/mass spectrometric analysis of selected post-translationally modified peptides : from fundamentals to shotgun proteomics
Authors
Issue Date2015
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Quan, Q. [全泉]. (2015). Multidimensional liquid chromatography/mass spectrometric analysis of selected post-translationally modified peptides : from fundamentals to shotgun proteomics. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5570800
AbstractThe continuing evolution of multidimensional liquid chromatography/mass spectrometry (MDLC–MS)–based proteomics is an important element of the developing field of shotgun proteomics for peptide sequencing, protein identification and quantification. The first part of this thesis, Chapter 2, demonstrates the development of a comprehensive automated MDLC platform capable of performing both quantitative proteomics analyses and post-translational modifications analysis—in particular, of protein tyrosine nitration and protein phosphorylation. The current multidimensional reversed-phase (RP) liquid chromatography design was employed with the addition of strong anion exchange (SAX) and cation exchange (SCX) columns. The inclusion of the complementary S(A/C)X column chemistries in the RP–SA(C)X–RP system allowed the retention of deprotonated peptides in the SAX trap column, followed by diversion of non-retained peptides to an online SCX trap column, thereby allowing identification of both anionic and cationic peptides from a single injection event. This MDLC RP–SA(C)X–RP platform provided more extensive protein and proteome coverage, thereby leading to improved protein quantification from analyses of Saccharomyces cerevisiae tryptic digests, a prototypical model proteome, as well as those of various other complex biological samples. Phosphorylated and 3-nitrotyrosyl–containing peptides—two important and biologically relevant post-translational modifications—were efficiently retained in this newly developed platform, in some cases without the need for any pre-enrichment steps. This RP–SA(C)X–RP technology performed well, as judged by the mapped protein inventory from the global collection of endogenous protein tyrosine nitration, the phosphoproteome, and its associated proteomics networks of permanent cerebral ischemia of Macaca fascicularis. The goal of the subsequent study was to gain insight into various aspects of the gas phase radical ion chemistry of phosphorylated peptides; these findings should provide an underlying scientific basis for the development of peptide sequencing strategies, because the general guidelines governing phosphorylated peptide radical cation dissociation remain poorly understood. No previous reports have described the successful generation of radical cationic phosphopeptides under low-energy collision-induced dissociation (CID). Chapters 3 and 4 describe a systematic investigation into the effect of the structures of the metal complexes on the efficient generation of radical phosphopeptide cations. To examine the mechanisms, energetics, and kinetics of these reactions, a combined experimental and computational approach was undertaken to facilitate a greater understanding of their dissociation behavior. Several model phosphopeptide radical cations were synthesized and characterized to formulate the fragmentation rules. The findings suggest that the dissociations of isomeric peptide radical cations can be more efficient than their isomerizations. In a situation similar to the dissociations of analogous even-electron protonated peptides, the losses of H3PO4 from both even- and odd-electron peptide cations are due preferentially to charge-driven mechanisms; the charge-driven loss of H3PO4 is favored as a result of the distonic radical character of the α-radical cation, enhancing the mobility of the charge carried by this species to generate structure-informative product ions.
DegreeDoctor of Philosophy
SubjectMass spectrometry
Liquid chromatography
Peptides
Dept/ProgramChemistry
Persistent Identifierhttp://hdl.handle.net/10722/219989

 

DC FieldValueLanguage
dc.contributor.authorQuan, Quan-
dc.contributor.author全泉-
dc.date.accessioned2015-10-08T23:12:17Z-
dc.date.available2015-10-08T23:12:17Z-
dc.date.issued2015-
dc.identifier.citationQuan, Q. [全泉]. (2015). Multidimensional liquid chromatography/mass spectrometric analysis of selected post-translationally modified peptides : from fundamentals to shotgun proteomics. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5570800-
dc.identifier.urihttp://hdl.handle.net/10722/219989-
dc.description.abstractThe continuing evolution of multidimensional liquid chromatography/mass spectrometry (MDLC–MS)–based proteomics is an important element of the developing field of shotgun proteomics for peptide sequencing, protein identification and quantification. The first part of this thesis, Chapter 2, demonstrates the development of a comprehensive automated MDLC platform capable of performing both quantitative proteomics analyses and post-translational modifications analysis—in particular, of protein tyrosine nitration and protein phosphorylation. The current multidimensional reversed-phase (RP) liquid chromatography design was employed with the addition of strong anion exchange (SAX) and cation exchange (SCX) columns. The inclusion of the complementary S(A/C)X column chemistries in the RP–SA(C)X–RP system allowed the retention of deprotonated peptides in the SAX trap column, followed by diversion of non-retained peptides to an online SCX trap column, thereby allowing identification of both anionic and cationic peptides from a single injection event. This MDLC RP–SA(C)X–RP platform provided more extensive protein and proteome coverage, thereby leading to improved protein quantification from analyses of Saccharomyces cerevisiae tryptic digests, a prototypical model proteome, as well as those of various other complex biological samples. Phosphorylated and 3-nitrotyrosyl–containing peptides—two important and biologically relevant post-translational modifications—were efficiently retained in this newly developed platform, in some cases without the need for any pre-enrichment steps. This RP–SA(C)X–RP technology performed well, as judged by the mapped protein inventory from the global collection of endogenous protein tyrosine nitration, the phosphoproteome, and its associated proteomics networks of permanent cerebral ischemia of Macaca fascicularis. The goal of the subsequent study was to gain insight into various aspects of the gas phase radical ion chemistry of phosphorylated peptides; these findings should provide an underlying scientific basis for the development of peptide sequencing strategies, because the general guidelines governing phosphorylated peptide radical cation dissociation remain poorly understood. No previous reports have described the successful generation of radical cationic phosphopeptides under low-energy collision-induced dissociation (CID). Chapters 3 and 4 describe a systematic investigation into the effect of the structures of the metal complexes on the efficient generation of radical phosphopeptide cations. To examine the mechanisms, energetics, and kinetics of these reactions, a combined experimental and computational approach was undertaken to facilitate a greater understanding of their dissociation behavior. Several model phosphopeptide radical cations were synthesized and characterized to formulate the fragmentation rules. The findings suggest that the dissociations of isomeric peptide radical cations can be more efficient than their isomerizations. In a situation similar to the dissociations of analogous even-electron protonated peptides, the losses of H3PO4 from both even- and odd-electron peptide cations are due preferentially to charge-driven mechanisms; the charge-driven loss of H3PO4 is favored as a result of the distonic radical character of the α-radical cation, enhancing the mobility of the charge carried by this species to generate structure-informative product ions.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.subject.lcshMass spectrometry-
dc.subject.lcshLiquid chromatography-
dc.subject.lcshPeptides-
dc.titleMultidimensional liquid chromatography/mass spectrometric analysis of selected post-translationally modified peptides : from fundamentals to shotgun proteomics-
dc.typePG_Thesis-
dc.identifier.hkulb5570800-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineChemistry-
dc.description.naturepublished_or_final_version-

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