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Mass Spectrometry – a Powerful Tool for Metabolomics Sandy Nargund Manager, MS & Chromato Shimadzu [Asia-Pacific] Pte Ltd

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Mass Spectrometry – a Powerful Tool for Metabolomics Sandy Nargund Manager, MS & Chromato Shimadzu [Asia-Pacific] Pte Ltd Mass Spectrometry – A Powerful tool for metabolomics Sandy Nargund Manager, MS & Chromato Shimadzu [Asia-Pacific] Pte Ltd 1 2 Biomarkers Identification and Validation 3 Mass Spectrometry –For Metabolomics GCMS-QP2010 Ultra GCMS-TQ 8040 LCMS-8060 Nexera UC- Online SFE/SFC iDPlus-Performance Bacterial Identification MALDI -7090 High Resolution TOF iMScope-Trio –Mass imaging 4 t Triple Quadrupole MS/MS Product ion scan t MRM- Multi Reaction Monitoring SIM (single analysis) MRM (MS/MS analysis) 50000 10 ppb 7500 10 ppb 40000 5000 30000 20000 2500 10000 0 0 1.0 1.5 2.0 2.5 min 1.0 1.5 2.0 2.5 min Eliminates background for High sensitivity but high trace-level quantitation with background high S/N GCMS- Gold Standard for Metabolomics 7 Metabolomics Research Using GC-MS/MS Metabolomics Research Discovery phase Validation phase Scan measurement MRM measurement MRM measurement (GC-MS/MS) (non-targeted analysis) (wide target analysis) (target analysis) Detect marker candidates Quantitate marker candidates and identify compounds with higher accuracy Accurate quantitation GC/MS Metabolite Database Ready to use method for quick start your Research 1. Method files Registered Measurement Number Derivatives Compounds Mode Registered 2. Library (scan) Organic acids, fatty Scan 428 3. Smart MRM database acids, amino acids, TMS sugars, etc. MRM 193 (automatic method creation tool) Scan 50 4. Instruction manuals Fatty acids Methylation MRM 50 Amino acids EZ:faastTM Scan 33 Easy Work Flow AART function for Automatic Adjustment of Retention Indices with just one injection Select components for measurement from the database. Smart MRM database Method is created Automatically Start acquisition. GCMS-TQ8040 1. Smart Productivity Analysis of 400 pesticides that used to require 2 or 3 methods, can now be accomplished in a single acquisition Three Smart functions improve analytical method by the new firmware protocol. productivity in your laboratory. 2. Smart Operation Smart MRM technology creates optimal MRM methods automatically. The “MRM Optimization Tool“ automates best MRM transitions for new compounds. 3. Smart Performance ASSP achieves high sensitivity at scan speeds of 20,000 u/second. Fastest MRM 800trans/sec. Single GC/MS mode with the maximum possible sensitivity and repeatability. Confidential 11 Advantages of GCMSMS Comparison of Reproducibility for Measuring Metabolites in Human Blood Serum Using MRM and Scan Modes 3-Hydroxyisovaleric acid-2TMS (100,000) (1,000) 7.5 131.10>73.00 247.00 247.10>73.00 205.00 2.0 5.0 %RSD (n=6) 2.5 1.0 Compound name MRM Scan 3-Hydroxyisovaleric acid-2TMS 3.99 14.0 15.0 15.5 16.0 15.0 15.5 16.0 Homocysteine-3TMS 5.04 23.4 Kynurenine-3TMS Aconitic acid-3TMS 5.98 N/A (10,000) (100) 4.0 307.10>218.10 7.5 424.10 Kynurenine-3TMS 6.48 24.5 307.10>192.10 307.10 3.0 5.0 2.0 2.5 1.0 0.0 43.5 44.0 43.5 44.0 44.5 MRM is able to eliminate the effects of interfering substances so that it can measure trace components accurately. 193 types of trimethylsilylated metabolites 50 types of fatty acid methyl esters (compatible with EI/PCI ionization) Advantage of GCMSMS Scan (Non-targeted) Discovery Scan/MRM data (x100,000,000) 2.25 2.00 1.75 1.50 1.25 1.00 0.75 MRM (Targeted) 0.50 0.25 Validation 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 Measurement of Scan/MRM mode Ex. TCA Cycle (SmartMRM) Scan section: Comprehensive search of metabolites MRM section: Accurate quantitation of target compounds Metabolites in urine using Scan/MRM Glutaric acid-2TMS (MRM) Malic Acid-3TMS Adipic acid-2TMS (MRM) 17.5 20.0 22.5 25.0 27.5 30.0 32.5 147 Measurement spectrum 233.00 Malic Acid-3TMS Scan 189.00 Measured 133 189 101 233 307 335 100 200 300 73 library spectrum 147 Library 133 233 101 189 307 335 21.25 21.50 21.75 22.00 100 200 300 Semi-quantitation Identification Metabolomics Solutions – Measurements to Pathway Analyses Scan Negative Negative Positive Positive Positive ID Name Serum001 Serum002 Serum003 Serum004 Serum005 GC/MS Metabolite 1 2-Aminoethanol-2T 1927822 1574800 1017918 1904832 1850236 2 Pyruvic acid-meto-T 10448 5442.353 9897.21 7413.618 8810.286 Database Ver. 2 3 Lactic acid-2TMS 147827 104531.8 109895.2 98769.1 88488.87 4 Glycolic acid-2TMS 216069 170218.9 210625.3 144679.6 152893.4 5 Alanine-2TMS 26855 20167.9 22414 17969.86 25549.17 6 2-Keto-isovaleric ac 71265 65720.31 63684.02 59946.26 51291.02 7 Glycine-2TMS 14620 10804.38 11295.5 9904.312 9789.166 8 2-Hydroxybutyric ac 35699 27065.86 27361.46 20173.2 30344.84 MRM Identify compounds Measure by GC-MS(/MS) Output data Enter PubChem ID Handbook From PubChem, link to Multivariate Analyze pathway KEGG (Detect marker analysis data candidates) Conventional Metabolomics M. W. Large LC-MS Peptide , Coenzyme, Nucleotide, Lipids Steroid, Vitamin Terpene Nucleoside , Sugar phosphate Hydrocarbo Sugar, Amino acids, Ester Organic acids, Fatty acids Ketone Alcohol Small GC-MS Volatility Non Volatility LCMSMS – Readymade Solutions LCMSMS- Readymade Solutions 18 Metabolites for major pathways in single method Ion pairing method (55 compounds) Non-ion pairing method (97 compounds) Glycolysis Glycolysis 2,3-Bisphosphoglyceric acid Adenosine 3',5'-cyclic Lactic acid 4-Aminobutyric acid Pyruvic acid Adenylsuccinic acid 3-Phosphoglyceric acid monophosphate Organic acids Argininosuccinic acid (2-Phosphoglyceric acid) Adenosine diphosphate 2-Ketoglutaric acid Cholic acid Creatine Dihydroxyacetone phosphate Adenosine monophosphate Aconitic acid TCA cycle TCA Citric acid Nicotinic acid Nucleotides Fructose 1,6-bisphosphate Adenosine triphosphate Fumaric acid Ophthalmic acid Glucose 1-phosphate Cytidine diphosphate Isocitric acid Orotic acid Malic acid Pantothenic acid Glucose 6-phosphate Cytidine monophosphate Succinic acid Taurocholic acid Pentose phosphate pathway Glycerol 3-phosphate Cytidine triphosphate Uric acid 4-Hydroxyproline Phosphoenolpyruvic acid Guanosine 3',5'-cyclic Alanine Adenine Pyruvic acid monophosphate Arginine Cytosine Fructose 6-phosphate Guanosine diphosphate Asparagine Guanine Aspartic acid Thymine Glyceraldehyde 3-phosphate Guanosine monophosphate Asymmetric dimethylarginine Uracil 6-Phosphogluconic acid Guanosine triphosphate Citrulline Xanthine Nucleotides, Nucleosides Erythrose 4-phosphate Coenzymes Thymidine diphosphate Cystine Adenosine Dimethylglycine Cytidine Ribose 5-phosphate Thymidine monophosphate Glutamic acid Guanosine Ribulose 5-phosphate Thymidine triphosphate Glutamine Inosine Glycine Thymidine Sedoheptulose 7-phosphate Uridine diphosphate Histidine Uridine Uridine monophosphate Homocystine Adenosine 3',5'-cyclic monophosphate Acetyl coenzyme A Standards Uridine triphosphate Isoleucine Adenosine monophosphate TCA cycle TCA Amino acids Leucine Cytidine 3',5'-cyclic monophosphate Succinyl coenzyme A Lysine Cytidine monophosphate NAD Methionine sulfoxide Guanosine 3',5'-cyclic monophosphate Guanosine monophosphate Alanine NADH Ornitine Phenylalanine Thymidine monophosphate Proline Arginine NADP Asparagine NADPH Serine FAD Symmetric dimethylarginine FMN Aspartic acid Threonine Coenzymes NAD Cysteine 2-Morpholinoethanesulfonic acid Tryptophan Transsulfuration pathway Methylationcycle Tyrosine 2-Aminobutyric acid Glutamic acid Methionine sulfone Valine Acetylcarnitine Amino acids Glutamine Cystathionine Acetylcholine Glycine Cysteine Allantoin Homocysteine Carnitine Histidine Methionine Carnosine Lysine 5-Glutamylcysteine Choline Citicoline Methionine Glutathione Oxidized glutathione Creatinine Phenylalanine S-Adenosylhomocysteine Cysteamine Serine S-Adenosylmethionine Dopa Others Dopamine Threonine Epinephrine Histamine Tryptophan Hypoxanthine Tyrosine Kynurenine Niacinamide Norepinephrine Standards 2-Morpholinoethanesulfonic acid Serotonin Ion pairing method contains metabolites in the central carbon Methionine sulfone metabolic pathway as target compounds and some amino acids and organic acids are added as target compounds in non-ion pairing method. Method Package for Primary Metabolites Ion pairing method Non-ion pairing method (55 compounds) (97 compounds) Glycolysis, TCA cycle (CoA), Amino acids, TCA cycle (organic acid), Target Pentose phosphate pathway, Bases, Nucleosides, Nucleotides, compounds Amino acids, Nucleotides Transsulfuration pathway, Methylation cycle LC Usage of ion pairing reagent Ion pairing reagent is not used conditions Separation by ODS column Separation by pentafluorophenylpropyl (PFPP) column Targeted Medical researcher in Medical researcher in pharma and Application pharmaceutical company and academia (ion pairing reagent is not academia allowed to use) Quality assessment by quantifying amino acids, organic acids etc. 57 Standards Mix: Ion Pair Method An aliquot of 57 STDs Mix was injected at a volume of 3 µL and separated using an ion-pairing chromatography. 60000 Tyrosine Glucose-6-phosphate Erythrose-4-phosphate TMP ADP 55000 MES (2-Morpholinoethanesulfonic Ribose-5-phosphate Ribulose-5-phosphate AMP TDP acid) Sedoheptulose-7- Pyruvate cGMP Phosphoenolpyruvate 50000 Glutamate phosphate CMP cAMP CTP Aspartate Fructose-6-phosphate NAD CDP GTP 45000 Phenylalanine Tryptophan Dihydroxyacetone- 6-Phosphogluconate UTP Glycerol-3-phosphate phosphate GDP ATP 40000 Glucose-1-phosphate UMP UDP TTP Glyceraldehyde-3- GMP 3-Phosphoglycerate NADPH 35000 phosphate /2-Phoshoglycerate 2,3-bis- 30000 NADP Phosphoglycerate Fructose-1,6-bis- Succinyl-CoA 25000 phosphate Acetyl-CoA NADH 20000 15000 Lysine Alanine Arginine Glutamine 10000 Histidine Threonine Glycine Cysteine 5000 Serine L-Methionine Asparagine sulfone
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