• Login
    View Item 
    •   Home
    • Massey Documents by Type
    • Theses and Dissertations
    • View Item
    •   Home
    • Massey Documents by Type
    • Theses and Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Peptide sequences by mass spectrometry : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

    Icon
    View/Open Full Text
    01_front.pdf (2.078Mb)
    02_whole.pdf (18.16Mb)
    Export to EndNote
    Abstract
    The preparation and mass spectrometry of permethylated peptide derivatives was investigated. Procedures for the modification of free peptides prior to permethylation were examined. Acetylation with methanol+ acetic anhydride was found to result in partial esterification of the peptide. Specific cleavage of the C-terminal residue was also observed; a mechanism is proposed for this reaction. Esterification with HCl in methanol followed by acetylation of the peptide ester gave a mixture of products due to random methanolysis during the esterification. Methods of acetylating free peptides were examined, and it was found that the use of water + acetic anhydride at room temperature resulted in rapid quantitative acetylation, with no significant side reactions. Reaction of an ethereal solution of diazomethane with the acetyl-peptide gave quantitative esterification with negligible byproduct formation. Use of dimethylsulfinyl sodium in dimethylsulfoxide, and methyl iodide for the permethylation of peptide derivatives was investigated. Suitable conditions were found for the preparation of the reagent and for its use in the permethylation reaction. Substitution at existing ester groups was found to occur during the permethylation, and the products were partially characterised. Use of the free acetyl-peptide rather than its methyl ester eliminated this side reaction. Introduction of more than the expected number of methyl groups was observed. This extra-methylation was found to occur mainly at specific residues, although some random methylation was observed. The conditions of permethylation were adjusted to minimise extra-methylation and limit it to specific sites in the molecule. Peptides containing aspartyl residues undergo chain cleavage; the products of this reaction were identified and a mechanism proposed for their formation. The permethylation reaction is discussed in relation to the formation of these artefacts; it is thought to involve deprotonation of the peptide to form a multiple anion. Reaction conditions are suggested to eliminate these side reactions. The mass spectrometry of permethylated peptide derivatives is discussed and the mass spectra of peptides of known sequence reported. The mass spectra show the sequence-determining fragments as the principal ions. This observation is rationalised in terms of the negative-inductive effect of the N-methyl groups. The simple procedure for interpreting the mass spectra of permethylated peptide derivatives is outlined, together with the use of minor fragmentation modes in identifying the molecular ion and sequencing peaks. Deuteriated methyl iodide, high resolution mass spectrometry and the detection of metastable transitions can all be used to confirm the deduced sequence. The techniques developed were applied to a mixture of free peptides isolated from cheese; the three peptides present were sequenced. The results were confirmed by high resolution mass measurement and permethylation with deuteriated methyl iodide. The present state of peptide sequence determination by mass spectometry is evaluated and possible future developments discussed.
    Date
    1970
    Author
    Kent, Stephen Brian Henry
    Rights
    The Author
    Publisher
    Massey University
    URI
    http://hdl.handle.net/10179/11100
    Collections
    • Theses and Dissertations
    Metadata
    Show full item record

    Copyright © Massey University
    Contact Us | Send Feedback | Copyright Take Down Request | Massey University Privacy Statement
    DSpace software copyright © Duraspace
    v5.7-2020.1
     

     

    Tweets by @Massey_Research
    Information PagesContent PolicyDepositing content to MROCopyright and Access InformationDeposit LicenseDeposit License SummaryTheses FAQFile FormatsDoctoral Thesis Deposit

    Browse

    All of MROCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Statistics

    View Usage Statistics

    Copyright © Massey University
    Contact Us | Send Feedback | Copyright Take Down Request | Massey University Privacy Statement
    DSpace software copyright © Duraspace
    v5.7-2020.1