Agilent Technologies, Inc. · NYSE:A
Agilent Technologies — Technical Presentation (November 2018)
34 slides from Agilent Technologies's technical presentation, Nov 7, 2018
Slide 1: Improved LC/MS Analysis of Polar Metabolites
Improved LC/MS Analysis of Polar Metabolites Emerging Omics Webinar Series Nov 7th 2018 Jordy Hsiao, Ph.D. R&D Scientist Agilent Technologies 1 November 2, 2018 For Research Use Only. Not for use in diagnostic procedures.
Slide 2: Current Methods for Metabolite Analysis
Current Methods for Metabolite Analysis • Polar metabolites are not retained in reversed phase Schematic of ion pairing*: chromatography • Could use ion pair reversed phase chromatography with reagents like tributylamine Ion-pair reagent • This improves retention and peak shape for polar acidic meta
Slide 3: New Zwitterionic HILIC Column for Metabolite Analysis
New Zwitterionic HILIC Column for Metabolite Analysis High organic mobile phase Based on Poroshell technology A A A 0.5µm H2O A H2O A H2O H2O H2O -HO - - - 1.7µm 2.7µm - 2 H2O H2O + + + + + Zwitterionic, high pH coating added Best performance for polar acidic metabolites 3 November 2, 2018 For Resea
Slide 4: Amino Acids
Amino Acids Ile Leu Low pH Positive Ion Mode Phe Arg Amino acids can be analyzed in either positive or negative ion mode. Val Gln Lys His Glu Ala Met Asn Trp Leu Asp Thr Tyr Ser Gly Ile High pH Negative Ion Mode Pro Gln Gly AspGlu • Low pH, positive ion is generally more Phe sensitive Val Ala Asn Ty
Slide 5: What About Other Analytes?
What About Other Analytes? TCA Cycle Some people need to analyze metabolites that present extra challenges: • Phosphorylated metabolites require negative mode MS and higher pH • Some acidic metabolites are sensitive to trace metal in LC flow path1-2 Glucose 1-phosphate Citrate Malate ATP 1 Myint KT
Slide 6: Initial Experimental Setup
Initial Experimental Setup LC/MS setup: Results: SS SS 1290 Binary Analytical 6545 5 ng LC pump Column QTOF Samples: • Phosphorylated Metabolites (e.g., ATP, sugar phosphates, etc.) 5 ng • Organic acids (e.g., malate, citrate, isocitrate, etc.) 6 November 2, 2018 For Research Use Only. Not for use i
Slide 7: Initial Column Testing Couldn't Detect Sub-10ng of Samples
Initial Column Testing Couldn’t Detect Sub-10ng of Samples Buffer: pH 6.8 ammonium acetate ATP 5 ng 50 ng 100 ng 250 ng 500 ng Stainless steel column PEEK-lined steel column 7 November 2, 2018 For Research Use Only. Not for use in diagnostic procedures.
Slide 8: Optimizing Analysis of Metabolites
Optimizing Analysis of Metabolites HDPE solvent bottles PEEK Capillaries Bio-Inert Quat Pump Bio-Inert Autosampler 8 November 2, 2018 For Research Use Only. Not for use in diagnostic procedures.
Slide 9: HILIC/MS Sensitivity Can Be Improved with PEEK-lined Capillaries
HILIC/MS Sensitivity Can Be Improved with PEEK-lined Capillaries Glucose-1-P/ AMP ADP ATP GTP NADP Glucose-6-P x10 5 -ESI EIC(346.0600) x10 5 -ESI EIC(426.0200) x10 4 -ESI EIC(505.9900) x10 2 -ESI EIC(521.9900) x10 3 -ESI EIC(742.0670) x10 5 -ESI EIC(259.0300) 1.25 1 1 6 1 1 6 0.8 Standard 0.75 0.75
Slide 10: But Not Everyone Has a Bioinert Pump!
But Not Everyone Has a Bioinert Pump! • Using a stainless steel 1290 binary pump • Buffer1: pH 9.0 ammonium acetate AMP ADP ATP GTP NADP G-1-P/G-6-P x105 -ESI EIC(346.0550) x105 -ESI EIC(426.0220) x104 -ESI EIC(505.9880) x104 -ESI EIC(521.9830) x104 -ESI EIC(743.0760) x105 -ESI EIC(259.0220) 3.0 1.5
Slide 11: Effect of Passivation on Chelating Organic Acids
Effect of Passivation on Chelating Organic Acids Succinate Malate Citrate/Isocitrate α-KG L-Glutamine Glutamate x105 -ESI EIC(117.0190) x105 -ESI EIC(133.0140) x106 -ESI EIC(191.0190) x105 -ESI EIC(145.014) x105 -ESI EIC(145.0600) x106 -ESI EIC(146.0460) 3.0 8 2.5 1.0 8 2.5 2.5 0.8 2.0 6 6 2.0 2.0 1
Slide 12: What Comes Next?
What Comes Next? This method has great performance for amino acids, sugars and phosphorylated compounds. However, you will have to passivate regularly if you are using a steel pump and steel autosampler flow path. . . . . and we still have problems with strongly metal-chelating organic acids Citrate
Slide 13: EDTA in Mobile Phase Solvents
EDTA in Mobile Phase Solvents A Succinate Malate Citrate/Isocitrate α-KG L-Glutamine Glutamate x105 -ESI EIC(117.0190) x106 -ESI EIC(133.0140) x106 -ESI EIC(191.0190) x105 -ESI EIC(145.0140) x105 -ESI EIC(145.0600) x106 -ESI EIC(146.0460) 6 1.0 1.5 4 6 5 3 0.8 4 3 1.0 2 4 0.6 3 2 0.4 2 0.5 2 1 1 0.2
Slide 14: Medronic Acid in Mobile Phase Solvents
Medronic Acid in Mobile Phase Solvents A Succinate Malate Citrate/Isocitrate α-KG L-Glutamine Glutamate x105 -ESI EIC(117.0190) x106 -ESI EIC(133.0140) x106 -ESI EIC(191.0190) x105 -ESI EIC(145.0140) x106 -ESI EIC(145.0600) x106 -ESI EIC(146.0460) 3.0 6 8 1.5 1.50 1.5 2.5 1.25 2.0 6 4 1.0 1.00 1.0 1
Slide 15: InfinityLab Deactivator Additive is Easily Cleared From the LC/MS System
InfinityLab Deactivator Additive is Easily Cleared From the LC/MS System x105 4 3 2 6th run with additive 1 1st post-additive run (no additive) 0 2nd post-additive run (no additive) 2 4 6 8 10 12 14 16 18 20 22 24 26 28 3rd post-additive run (no additive) Counts vs. Acquisition Time (min) 4th post-a
Slide 16: LOD of SSTL and PEEK-lined Columns on 6490 QQQ
LOD of SSTL and PEEK-lined Columns on 6490 QQQ SSTL PEEK-lined With With SSTL PEEK-lined Medronic Acid Medronic Acid ATP x104 -ESI MRM (505.9885 -> 158.7) x105 -ESI MRM (505.9885 -> 158.7) x10 5 -ESI MRM (505.9885 -> 158.7) x10 5 -ESI MRM (505.9885 -> 158.7) 1.0 1.0 1.0 2 5 ng 1 0.5 0.5 0.5 (5 ppm)
Slide 17: Improved Run to Run Reproducibility with the Additive
Improved Run to Run Reproducibility with the Additive A Succinate Malate Citrate/Isocitrate AMP ADP ATP x10 5 -ESI EIC(117.0190) x10 6 -ESI EIC(133.0140) x10 6 -ESI EIC(191.0190) x10 5 -ESI EIC(346.0550) x10 5 -ESI EIC(426.0220) x10 4 -ESI EIC(505.9880) 3 8 3 1.5 4 4 2 6 2 1 Control 2 1 2 1 0.5 4 2
Slide 18: Salt Tolerance Study
Salt Tolerance Study Upper Range of a Human Urine Sample Major Urine [Concentration] [mM] Components Urea 23.3 g/L 390 mM A 4M Urea, 2M NaCl stock solution was made for salt spike-in experiment. Chloride 8.4 g/L 236 mM Sodium 4.4 g/L 191 mM pH 6.3 G-1-P/G-6-P AMP ADP ATP GTP NADP x10 5 -ESI EIC(259.
Slide 19: Outline
Outline Brief Introduction • Background • Current methods • Challenges Story of the InfinityLab Poroshell 120 HILIC-Z column • Discovery of a novel mobile phase additive Applications • Metabolomics analysis of MTX-treated cells • Monitoring metabolites from white blood cells (USC collaboration) • Mo
Slide 20: Metabolomics Analysis of MTX-treated Cancer Cells
Metabolomics Analysis of MTX-treated Cancer Cells Methotrexate inhibits dihydrofolate reductase (DHFR) and thereby inhibits the synthesis of folate -> DNA, RNA, and proteins Cells were spun down, Extraction solution washed and collected (2:2:1 MeOH:ACN:H2O with 0.1% formic acid) 5 µM MTX-treated for
Slide 21: MTX Induces Fructose 1,6-bisphosphate and Adenosine Levels
MTX Induces Fructose 1,6-bisphosphate and Adenosine Levels • Exogenous administration of F-1,6-P2 has been shown to sustain glycolysis and increase ATP production1-3. • Methotrexate systematically generates extracellular adenosine and subsequent activation of adenosine receptor A2a4. 1 Planas ME et
Slide 22: Analysis of Polar Metabolites Extracted from White Blood Cells
Analysis of Polar Metabolites Extracted from White Blood Cells Drs. Jonathan Katz and Sujatha Chilakala Sample Information Analytical Method • WBCs extracted from whole blood of Mobile phase: two volunteers using density gradient A: 90/10 Water/ACN, 10 mM Ammonium formate, pH 9.2, 5 µM additive • Po
Slide 23: Analysis of Polar Metabolites Extracted from WBCs: ATP
Analysis of Polar Metabolites Extracted from WBCs ATP Control With Additive 23 November 2, 2018 For Research Use Only. Not for use in diagnostic procedures.
Slide 24: Analysis of Polar Metabolites Extracted from WBCs: Malate
Analysis of Polar Metabolites Extracted from WBCs Malate Control With Additive 24 November 2, 2018 For Research Use Only. Not for use in diagnostic procedures.
Slide 25: Analysis of Polar Metabolites Extracted from WBCs
Analysis of Polar Metabolites Extracted from WBCs Molecular feature extraction MFE Score With additive(1424) No additive(1417) 100 677 540 99.9-90 264 196 89.9-80 246 163 79.9-70 237 518 25 November 2, 2018 For Research Use Only. Not for use in diagnostic procedures.
Slide 26: Why is Cell Media Analysis Critical?
Why is Cell Media Analysis Critical? Example: Optimize tyrosine concentration to Example: Monitoring excreted lactate increases recombinant protein production concentration and effects on cell growth [Lactate] + Tyr 0 1 2 3 4 5 6 Days Tyr x10 7 Lac 2 1 0 1.4 1.6 1.8 2 2.2 2.4 26 November 2, 2018 For
Slide 27: Experimental Workflow
Experimental Workflow 27 November 2, 2018 For Research Use Only. Not for use in diagnostic procedures.
Slide 28: Monitoring Secreted Metabolic Waste in Cell Culture Media
Monitoring Secreted Metabolic Waste in Cell Culture Media α-D-Glucose/ Lactate β-D-Glucose Malate α-KG Glutamate Citrate x107 -ESI EIC(89.0240) x106 -ESI EIC(179.0561) x104 -ESI EIC(133.0140) x104 -ESI EIC(145.0140) x104 -ESI EIC(146.0460) x105 -ESI EIC(191.0190) 2 6 2 2 2 4 4 1 Day 0 1 1 1 2 2 0 0
Slide 29: Monitoring Consumed Nutrients in Cell Culture Media
Monitoring Consumed Nutrients in Cell Culture Media Serine Valine Threonine Leucine/Isoleucine L-Glutamine Lysine x105 -ESI EIC(104.0353) x105 -ESI EIC(116.0717) x105 -ESI EIC(118.0510) x106 -ESI EIC(130.0873) x106 -ESI EIC(145.0600) x104 -ESI EIC(145.0980) 4 3 7.5 2 1.0 2 5.0 1 2 1 Day 0 1 2.5 0.5
Slide 30: Monitoring Consumed Nutrients in Cell Culture Media
Monitoring Consumed Nutrients in Cell Culture Media Methionine Histidine Phenylalanine Arginine Tyrosine Tryptophan Cystine x105 -ESI EIC(148.0430) x105 -ESI EIC(154.0620) x105 -ESI EIC(164.0720) x104 -ESI EIC(173.1040) x105 -ESI EIC(180.0660) x104 -ESI EIC(203.0830) x104 -ESI EIC(239.0166) 3 7.5 1.
Slide 31: Monitoring Spent Culture Media by HILIC-LC/MS
Monitoring Spent Culture Media by HILIC-LC/MS Glucose Consumption and Secreted Metabolites Consumed Nutrients 120 120 100 100 80 80 % Change % Change 60 60 40 40 20 20 0 Day 0 Day 1 Day 2 Day 3 Day 6 0 Serine Valine Threonine Leucine Day 0 Day 1 Day 2 Day 3 Day 6 Isoleucine L-Glutamine Lysine Methio
Slide 32: Summary
Summary • A new zwitterionic HILIC column was developed to improve the separation of polar metabolites. LCGC article Analytical Chemistry Application Note (June 2018) – HILIC-Z (July 2018) - Additive 5994-0024EN • An optimized method was developed to address poor peak shapes and low sensitivity for
Slide 33: Acknowledgements
Acknowledgements Agilent USC collaborators • Ta-Chen Wei • Jonathan Katz • Andrew Kennedy • Sujatha Chilakala • Jun Li • Hongfeng Yin • Oscar Potter • Alex Apffel • Genevieve C. Van de Bittner • Chris Miller • Steve Fischer • Adam Bivens • Fred Chan • Te-Wei Chu • Greg Staples 33 November 2, 2018 Fo
Slide 34: November 2, 2018 For Research Use Only. Not for use in diagnostic procedures.
34 November 2, 2018 For Research Use Only. Not for use in diagnostic procedures.
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