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    Plasma nervonic acid levels were negatively associated with attention levels in community-living older adults in New Zealand
    (Springer Nature, 2022-08) de Seymour JV; Beck KL; Conlon CA; Von Hurst PR; Mumme KD; Haskell-Ramsay CF; Jones MB
    The global population is aging. Preserving function and independence of our aging population is paramount. A key component to maintaining independence is the preservation of cognitive function. Metabolomics can be used to identify biomarkers of cognition before noticeable deterioration. Our study investigated the plasma metabolome of 332 community-living New Zealanders between 65 and 74 years of age, using gas chromatography-mass spectrometry. Six cognitive domains were assessed. Of the 123 metabolites identified using an in-house mass spectral libraries of standards, nervonic acid had a significant, inverse association with the attention domain (P-value = 1.52E− 4; FDR = 0.019), after adjusting for covariates (apolipoprotein E -ε4 genotype, sex, body fat percentage (standardised by sex), age, education, deprivation index, physical activity, metabolic syndrome, polypharmacy, smoking status, and alcohol intake) and multiple testing. Attention is defined as the ability to concentrate on selected aspects of the environment while ignoring other stimuli. This is the first study to identify nervonic acid as a potential biomarker of attention in older adults. Future research should confirm this association in a longitudinal study.
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    Peptide fingerprinting and predictive modelling of fermented milk : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North Campus, New Zealand
    (Massey University, 2021) Murphy, Fionnuala
    Fermented milk products are valued by consumers and the food industry for their nutritional properties, pleasant taste, and texture. Consumer demands and expectations for such products are constantly changing. Understanding how consumers perceive the sensory characteristics of food and the relationship these characteristics have with the chemical components of food can provide insight that can enable food researchers and manufacturers to develop food products that are tailored to provide enhanced sensory qualities. Establishing techniques that allow for in-silico prediction or correlation of sensory qualities can enable a more rapid approach that would aim to enable researchers to meet the demands of consumers. This research firstly explored mass spectrometric techniques for the rapid fingerprinting of milk and fermented milk products, using Matrix-Assisted Laser Desorption Ionisation - Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and Rapid Evaporative Ionisation Mass Spectrometry (REIMS), two technologies that require minimal sample preparation and can rapidly generate a fingerprint of a food’s chemical components. Peptide fingerprints obtained by MALDI-TOF MS and analysed by principal component analysis were effective at discriminating the two fermented milk and milk samples. Supervised discrimination of low molecular weight fingerprints obtained via REIMS and MALDI-TOF MS proved less effective but demonstrated some potential and could be used alongside other analyses in future studies. These techniques were explored with a view to establishing a technique that could provide rapid insights into a food’s chemical composition, and which could also effectively discriminate the chemical components of the product. Such techniques could be used for rapid screening of products and can provide insight into the chemical components that are driving the variation in different products, which may be reflective of the differences in sensory characteristics. Next, peptide fingerprinting and predictive modelling were investigated in milk fermented with various bacterial combinations, including probiotic cultures. Fingerprinting was performed on samples collected at each hour of fermentation. Predictive modelling techniques, using both regression and classification approaches, were trialled in order to predict the change in signal intensity throughout fermentation. This aimed to understand if peptides could be predicted throughout fermentation, with a view to enable the targeted prediction of desirable peptides, or other relevant components, which may impart favourable sensory qualities in the final product. Regression techniques were somewhat effective for predicting the signal intensity of individual m/z ions throughout fermentation. Most of the ions did not follow a linear relationship, and, as such, a multiple linear regression model was unable to model most of the ions. Using a generalised additive model, a non-linear approach, improved the performance in most cases and could predict the signal intensity of individual ions throughout fermentation. However, the model was unable to correctly predict all cases. Classification techniques were effective for predicting the general direction of the signal intensity between start and end fermentation times. Five classification techniques were trialled, with each model providing accurate predictions for the increase or decrease of signal intensity between early and late fermentation times. Lastly, consumer panellists were recruited to evaluate the change in important sensory characteristics throughout the fermentation of milk prepared using two different starter cultures. This aimed to understand if consumer responses to such products could be correlated with instrumental analysis, in order to predict the consumer responses from instrumental data. Consumers perceived significant differences in bitterness and flavour intensity between fermented milk samples at different fermentation time points. There were significant correlations between peptide fingerprints and the consumer rankings for the sensory attributes in each fermented milk product. XGBoost regression could predict consumer responses with reasonable accuracy. This thesis explored the fermentation of milk using specific bacteria and fermentation processes. To validate this work, further products could be explored, in addition to different processing parameters. Furthermore, a more in-depth analysis of the chemical components of the products could be investigated and analysed with additional sensory evaluation to further explore and confirm the findings.
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    Mass spectrometry-based metabolomics as a tool for biomarker discovery and diagnosing metabolic health : a thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science, School of Health Science, Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Zhanxuan, Wu
    Although obesity and prediabetes have been long-established risk factors for type 2 diabetes (T2D), excess fat deposition in visceral and ectopic organ sites (i.e. the “risky” fat depot) has been increasingly recognised as key conduits for T2D. However, quantification of these fat depots relies on expensive and time-consuming imaging techniques. There is a need for identification of biomarkers predictive of risky fat depot levels. Metabolomics is a promising tool for discovering novel markers and generating mechanistic speculation. This PhD project aims to identify and understand plasma metabolite markers associated with metabolic risk factors including elevated fasting plasma glucose (FPG), fat deposition in visceral (VAT) and ectopic organ sites (liver and pancreas) in non-T2D human. To achieve this, a workflow to select the optimal injected concentration of different sample types for two complementary LC-MS untargeted analyses (polar metabolites and lipidomics) was established and applied to examine the value of measuring the plasma metabolome as a proxy for metabolite concentrations in various tissue sites including adipose tissue, muscle and the liver. Lastly, plasma metabolomic signatures for elevated FPG and VAT were characterised and metabolite markers predictive of VAT and ectopic fat deposition in the liver and pancreas were identified. This PhD study highlighted the critical importance in optimising injected concentration for LC-MS analysis of different sample types to ensure the maximal number of linear features were obtained, and for the first time showed the plasma metabolomics profile was more reflective of the liver profile than muscle or adipose tissue. Subsequent metabolomics characterisation of clinical plasma samples reported profound associations between FPG or VAT with changes in several glycerolipid species independent of gender, ethnicity, age and body mass index (BMI). VAT was additionally associated with changes in phospholipid, ether-linked phospholipid and sphingolipid species independent of covariates. Liver fat deposition was predicted by a number of glycerolipid, phosphatidylethanolamine and dihydroceramide lipid species whose plasma concentrations were linearly correlated with the liver counterparts. A novel marker, sulfolithocholic acid, for the prediction of pancreatic fat independent of age, BMI and visceral adiposity was also identified. Finally, the study also reported an improved prediction of ectopic fat deposition by utilising a panel of metabolite markers compared to clinical measurements, and demonstrated the usefulness of the metabolomic signature to identify a subset of normoglycaemic individuals with a worse cardiometabolic profile. Findings from this PhD study highlighted the value of metabolomics as a promising tool to capture metabolic risk, and that candidate markers identified by metabolomics may offer opportunities for improved risk prediction and stratification, disease progression monitoring and to develop alternative means for the measurement of the effectiveness of dietary interventions.
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    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
    (Massey University, 1970) Kent, Stephen Brian Henry
    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.
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    Application of electrospray mass spectrometry to the analysis of lipids : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Food Technology at Massey University
    (Massey University, 2000) Norris, Carmen
    This work investigated the application of electrospray mass spectrometry (MS) to the elucidation of lipid structure, especially related to milkfat. This was the first time that the instrument in our laboratory had been used for this purpose. Standard samples of triacylglycerols (TAGs) were prepared in 1,2 dichloroethane: acetonitrile:formic acid (63:35:2) at approximately 0.005 mg/mL and were then used to optimise and quantify the ion signal (response) generated by the Perkin Elmer Sciex API300 electrospray mass spectrometer. Both MS and tandem MS experiments were performed. In the spectra, the TAG molecule was seen as an ammonium ion adduct (M+NH4)+. It was found that the relative responses of diacylglycerol (DAG) ions, formed during front end fragmentation of the (M+NH4)+ ion, depended on the position of the 'lost' fatty acid on the glycerol backbone and its carbon number, with the former rather than the latter being the more critical. This information enabled the fatty acid esterified to the sn2 carbon of the glycerol backbone to be identified, and also demonstrated that it was possible to identify each of the fatty acids in a TAG molecule accurately by molecular and DAG ion identification. MS/MS experiments were performed on DAG ions, rather than parent ions, to identify and measure the response of acylium ions generated during collision-assisted dissociation (CAD). In contrast to the response of the DAG ions above, it was found that the response of these acylium ions was dependent on their carbon number and degree of saturation rather than the position that the fatty acid had held on the glycerol backbone. Optimal voltage settings for analysis of TAGs by infusion MS were obtained, which gave good quality spectra and ample amounts of molecular and DAG ions. With this information, a novel liquid chromatography-mass spectrometry (LC-MS) method, which was able to characterise the TAGs in a number of complex lipid samples, was developed. The method was used to elucidate differences in the TAG structure of different bovine milkfats and also differences in fat from milk of various mammalian species.
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    Proteins isolated with TRIzol are compatible with two-dimensional electrophoresis and mass spectrometry analyses
    (Elsevier Masson, 2012) Young C; Truman P
    TRIzol is used for RNA isolation but also permits protein recovery. We investigated whether proteins prepared with TRIzol were suitable for two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectrometry. Proteins from TRIzol-treated SH-SY5Y cells produced 2-DE spot patterns similar to those from an equivalent untreated sample. Subsequent identification of TRIzol-treated proteins using peptide mass fingerprinting was successful. TRIzol exposure altered neither the mass of myoglobin extracted from sodium dodecyl sulfate (SDS) gels nor the masses of myoglobin peptides produced by in-gel trypsin digestion. These findings suggest that proteins isolated with TRIzol remain amenable to proteomic analyses.