LC MS1 - Mass Spectrometry Analysis of Small Molecules...

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1 Mass Spectrometry Analysis of Small Molecules Metabolomics - A realm of small molecules (<1000 Da) Coined in the 1990s: Oliver, S. G., Winson, M. K., Kell, D. B. & Baganz, F. (1998). Jeevan Prasain, PhD What is metabolomics? Identification and quantification of the complete set of metabolites in a biological system Quantitative measurement of the dynamic metabolite response of living systems to pathophysiological stimuli or genetic modification provides important insights into physiological and disease states and facilitate in depth understanding of underlying biochemical pathways. Metabolomics has the potential to provide valuable information on various patho-physiological conditions such as cancer, Alzheimer’s and cardiovascular diseases.
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2 Key applications Toxicity assessment/toxicology Functional genomics Nutrigenomics Herbal formulation, quality control and clinical trial . Metabonomics-NMR as analytical plateform Prior to LC-MS based metabolomics, the small molecule -ome was analyzed by 1 H- and a lesser extent 13 C-NMR (Metabonomics) NMR is carried out on urine and plasma Does not require chromatography, but sample has to carefully dried and redissolved in 2 H 2 O Compounds analyzed by their chemical shifts Assisted by very high magnetic fields to obtain resolution Not very sensitive, but microliter probes now in use
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3 Genome Transcriptome Proteome Metabolome Phenotype What might be happening in a cell Snapshot of the entire physiology Amino acids Fatty acids Phenolics Prostaglandins Steroids Organic acids Organic amines Nucleosides Nucleosides Polyamines Organic acids Lipids • Profiling involves finding of all metabolites detectable to a selected analytical technique with statistically significant variations in abundance within a set of experimental and control groups. •Identification of chemical structures of metabolites of interest after profiling •Quantification and validation •Interpretation of data making connections between the metabolites discovered and the biological conditions Steps involved in metabolomic analysis Possible metabolites Metabolomics in the context of other omics Metabolomics is complementary to the other -omics and the combination of these three may provide important information about the status of a cell Workflow for metabolome analysis Sample collection Treatment or Diseased group Control group Urine, plasma, tissue etc. Sample preparation Internal standard spiking for quantitative analysis Extraction (liquid-liquid, ppt with or without hydrolysis) Sample analysis LC-MS and LC-MS/MS analysis • Q1 scan using HPLC or UPLC/-TOFMS • +ve/-ve ion mode- accurate mass measurement • MS/MS experiments using a hybrid instrument Q-Trap Data analysis Multivariate analysis, e.g. PCA Data export Marker identification
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4 Points to be considered in LC-MS analysis Choice of ionization mode - ESI vs. APCI +ve/-ve modes Choice of eluting solvent - methanol vs. acetonitrile
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LC MS1 - Mass Spectrometry Analysis of Small Molecules...

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