Abstract
Problem statement: Comparison and validation analysis of the conventional and power-adjusted Microwave-Assisted (MA) techniques of TMS derivatization using Gas Chromatography-Mass Spectrometry (GC-MS) and Multivariate Analysis (MVA) of Partial Least Squares Analysis (PLS-DA). Further improvement of the conventional technique using vigorous shaking was tested and analyzed. Approach: Cross-validation and response permutation test of PLS-DA and S-plot of Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) was applied to the extracellular data of Lactococcus lactis, which was analyzed by GC-MS. The analyzed samples were firstly derivatized using Methoximination (MeOX) and N-methyl-N-trimethylsilyl fluoroacetamide (MSTFA) followed by conventional and power-adjusted Microwave-Assisted (MA) heating treatment. Results: The supervised PLS-DA applied to extracellular data failed to show the same clustering results between conventional and power-adjusted Microwave-Assisted (MA) techniques. It was suggested that the type of heating used in the derivatization techniques had affected the detection of groups of metabolites. Furthermore using the UV-scaling method, S-plot and Variable Importance for Projection (VIP), about 40 metabolites that were responsible towards the clustering and separation showed in PLS-DA score plots were successfully indicated. Conventional technique with vigorous shaking showed clear clustering according to groups compared to MA technique. Conclusion: Type of heating applied to the TMS derivatization showed effects towards the detection of metabolites where conventional technique indicated strong clustering compared to MA technique.
Highlights
Microbial metabolomics focusing on the measurement of low molecular mass metabolites is routinely used to screen metabolites that were indicative of specific regulator and mechanism
Several improvements have been described to irradiation, we focused on the usage of MSTFA to shorten the duration of heat treatment and increased analyze metabolites extracted from L. lactis extracellular grown in condition at 30-37°C and TMS derivatization: The derivatization technique used in agitation condition
Due to inconsistency and poor separation obtained by Principal Component Analysis (PCA), supervised PLS-DA was carried out by fitting each replicates tested for its fermentation conditions
Summary
Microbial metabolomics focusing on the measurement of low molecular mass metabolites is routinely used to screen metabolites that were indicative of specific regulator and mechanism. Microwave irradiation as a heating treatment for with current separation technologies, methoximation and silylation processes prior to GC-MS development of multiple techniques and methods that analysis has been acknowledged and widely used in focused on the single analytical instrument to quantify almost all biological samples including environmental and profile all metabolites is possible. (Kamenik et al, 2010; Varghese et al, 2010) It is an Generally, the facultative anaerobic and mesophilic excellent high-throughput analytical tool that offers L. lactis produces a mixture of metabolites including reproducibility, high resolution and relatively cheap amino acids, organic acid, fatty acid, alcohols, compared to other analysis tools (Park et al, 2010). The TMS derivatization usually The supervised PLS-DA is useful to visualize high comprises of a long heating treatment at 37-40°C with dimensional data, perform discriminant analysis and two steps; methoximation using methoxyamine identify relevant potential metabolites involved in hydrochloride dissolved in pyridine and silylation using metabolic changes.
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