HPLC-MS/MS and Metabolomics

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1 7/9/18 HPLC-MS/MS and Metabolomics MATTHEW BERNIER, PH.D. CCIC MS&P SUMMER WORKSHOP JULY 18 Thanks to Yu Cao Importance of the metabolome Applications of metabolomics in cancer research Kathleen A. Vermeersch and Mark P. Styczynski J. Carcinogenesis, 13, 1, doi: 1.13/ Encompasses biomolecules intracellular and extracellular that aren t proteins/nucleic acids Lipids (Lipidomics) Carbohydrates (Glycomics) Small peptides (Peptidomics) Other small molecules Single Amino Acids Cytokines Phosphates Many many more Publications on Metabolomics Number of publications per year Search engine: Web of Science Topic metabolomics 3 Why LC ESI-MS for Metabolomics? A feature is simply a signal that relates to one unique m/z and retention time (for MS) 5 # of hits Year Metabolomics short course at ASMS 1 Basic Metabolomics Workflows Metabolomics: the apogee of the omics trilogy Gary J. Patti, Oscar Yanes and Gary Siuzdak Molecular Cell Biology, 1, 13, Sample Preparation 1

2 7/9/18 Sample Preparation Sample prep often most consuming (difficult) step Sample prep/clean-up separates interfering species from analyte Example: analysis of drug and metabolites in plasma need to remove protein interferences Off-line or in-line from MS/MS detection Sample prep/clean-up to concentrate analyte Example: Pesticides in drinking water Basic principle of sample prep involves preferential binding of analyte over interfering species or vice versa, followed by elution to MS or MS/MS analysis Separation technologies essential in sample prep General Considerations when Dealing with samples for MS or MS/MS Ionization method? Instrument availability Sensitivity/Quantification Time of analysis In vivo sample pre-ms treatment? Chromatographic/extraction methods: the shorter, the better GC, HPLC, zip-tip, solid phase extraction (SPE), dialysis, etc. Derivatization: the simpler, the better to increase volatility (GC); to study neutral loss; to increase ionization efficiency Types of Separation Technologies Method Separation based on Separation done using Further steps Liquid-liquid Extraction Partitioning in one of two liquid phases Glassware Liquid-liquid Extraction An immiscible solvent is added to the sample which then separates into distinct liquid phases. Some sample analytes will go into the bottom phase (Aqueous), some will separate into the top phase (Organic) Large solvent consumption Time/labor intensive May need evaporation step > 1 extraction if mixture of analytes Emulsions and contamination issues faculty.ksu.edu.sa Types of Separation Technologies Solid Phase Extraction Method Separation based on Separation done using Further steps Liquid-liquid Extraction Solid-phase Extraction Partitioning in one of two liquid phases Adsorption/ partitioning onto solid sorbent Glass ware Cartridges, disks, filters, plates Uses chromatographic particles Packed-bed column cartridges or similar Well established commercial technology (1978) 1s of literature references Clean extracts Good recovery for polar analytes Sample must be in liquid state Driving force: gravity, pressure, vacuum Automation possible cartridges 96 well plate disk

3 7/9/18 Solid Phase Extraction Types of Chromatography Normal Phase Non-polar mobile phase Polar stationary phase Reversed Phase (Most common) Polar mobile phase Non-polar stationary phase Ion Exchange Buffer/Ionic mobile phase Cationic/Anionic exchange stationary phase Manufacturer Waters Varian Baker 3M Supelco + Many Others Brand Name SEP-PAK OASIS BondElute BakerBond Empore Supelclean Solid Phase Extraction- common protocol Procedure: Sample Prepare: Homogenize, suspend, centrifuge, etc Load onto conditioned cartridge Wash off weakly retained interferences with weak solvent Elute product with strong solvent Analyze: HPLC, GC-MS, LC-MS/MS Solid Phase Extraction Example (KNO 3 ) n K + a) pure analyte (control) Types of Separation Technologies Method Separation based on Separation done using Further steps b) engine oil contaminated parking lot oil Liquid-liquid Extraction Partitioning in one of two liquid phases Glass ware Solid-phase Extraction Adsorption/ partitioning onto solid sorbent Cartriges, disks, filters, plates c) same as b) after organic matter removal by SPE Dialysis/Ultrafiltration Molecular weight/size SlideAlyzer/tubing Gapeev, A. and Yinon, J. J. Forensic Sci., 9 Dialysis Tubing or Slide A-Lyzer (Different MWCO Ranges.1.5 ml capacity Sample loading here Types of Separation Technologies Useful for biologicals Method Separation based on Separation done using Further steps Spin filters Polyethersulfone membrane (Vivaspin, ex) volumes from 1 μl to ml, with a range of molecular weight cutoff values from Mr = 3-1 Liquid-liquid Extraction Solid-phase Extraction Dialysis/Ultrafiltration Precipitation Partitioning in one of two liquid phases Adsorption/ partitioning onto solid sorbent Molecular weight/size Solubility Glass ware Cartriges, disks, filters, plates SlideAlyzer, tubing, spin filter 3

4 Whole blood 7/9/18 Blood Plasma and Serum (~8% of metabolomics studies) Anti-coagulant Choice - Effects anti-coagulant (EDTA or heparin or citrate) unclotted blood 6% in metabolomics Plasma collection can be more reproducible More proteins Anticoagulants may affect separation and detection Example: commercially available rat plasma Lithium Heparin significant loss of features in the 5 65-s region in negative ESI mode Wedge, (11) Anal. Chem. EDTA peak natural process of clotting at room temperature, 3-6 min clotted blood DajanaVuckovic (1) Anal BioanalChem, 3, 153 % in metabolomics Possible enzymatic degradation Possible loss of some metabolites during clot large peak with a retention time of ~1 min cause severe ionization suppression Pereira, (1) Metabolomics Citrate peak severe suppression of the underneath peak using both reversed-phase HPLC and HILIC Vuckovic, (11) Anal. Chem. adapted from Junhua Wang, Ph.D. at Thermo adapted from Junhua Wang, Ph.D. at Thermo Plasma and Serum - Deproteinization 1:3 plasma: solvent (v/v) 1: to1:5 used in literatures msquan/prep.htm 95% water Urine (7-8 % of metabolomics studies) Relatively protein free urea, creatinine and uric acid typically present as products of nitrogen metabolism and needed to be removed from the body urea present at up to % No, still a lot of proteins! If you dry down, you will see them! salts Na, Cl, K, PO, SO 3 I, NH 3 Protein removal efficiency Metabolite coverage ph -8, meat diets tend to produce a lower ph than vegetarians methanol (98 %) ethanol (96 %) acetonitrile (9 %) High-56 Higher-1919 Medium-166 other metabolites typically lower MW species such as amino and organic acids and lower concentrations of high MW lipids (cf serum) Want, Siuzdak (6) Anal. Chem. 78:73 acetone/methanol (95 %) heat and acid treatment (98 %) High-136 Low-9 A relatively simple biofluid Actually detect many species that are different from blood. adapted from Junhua Wang, Ph.D. at Thermo Urine LC/MS Analysis Protocols Collection Lyophilization Dilution and centrifugation (high water) Recommend: using 5k MWCO Dilution and centrifugation (high organic) RP-LC-MS analysis HILIC-LC-MS analysis Cerebrospinal Fluid (CSF) Brain contains about 16 ml on average.8% proteins Water Inorganic salts Metabolites including neurotransmitters and GABA, glutamate, glutamine etc Wishart, (8) J Chromatogr B. The human cerebrospinal fluid metabolome (~% of metabolomics studies) Gika, (8) J. Sep. Sci. Hydrophilic interaction and reversed-phase ultra-performance liquid chromatography TOF-MS for metabonomic analysis of Zucker rat urine. Callahan, (9) J. Sep. Sci. Profiling of polar metabolites in biological extracts using diamond hydride-based aqueous normal phase chromatography. Collect, typically a lumbar puncture, freeze and store at -8ºC (1) Quench the metabolism; () prepare and analyze as for serum/plasma!

5 7/9/18 Mammalian Tissues Tissues are an ensemble of interacting cells Blood presence in tissue, influences metabolome of tissue! Different types of tissues nervous tissue, central nervous system (brain, spinal cord, periphereal nerves) epithelial tissue, layers of cells that cover organs such as skin muscle tissue, smooth, skeletal and cardiac, produces force and causes motion connective tissues are fibrous tissues (~5% of metabolomics studies) Mammalian Tissue Extraction 1- mg of tissue extracted Extraction typically involves homogenisation of tissue using a homogeniser or a shaker with stainless steel balls manual with a mortar and pestle in liquid nitrogen Typically perform a modified Folch extraction methanol/water/chloroform two phases collected (polar and non-polar) Wu, (8) Anal Biochem. 15;37(): Lyophilisation, grinding and extract powder is a second option Bobeldijk, (8) J Chromatogr B, 871():36 Wheaton Glass Tissue Grinder. ml, minimal sample loss Mammalian Tissues: Folch Extraction Collect and freeze immediately (1-mg) Solvent Mixtures for Tissue Extraction Thaw on ice and wash in cold saline ACN/MeOH Homogenise in appropriate solvent at ºC (::1 methanol:chloroform:water) Shake at C 163 x 6 x 76 x Rats eyes for retinal degeneration disease. Add 1:1 chloroform:water Centrifuge, two phase supernatant (chloroform at bottom) 57 x 17 x infinity Metabolomics Approach Studies the Metabolic Perturbation Induced Molecular Changes in Retinal Degeneration Disease Transfer two phases to separate tubes for analysis Wang, Siuzdak et al adapted from Junhua Wang, Ph.D. at Thermo Cell Line Samples Preparations Wash (fast <1s, physiological buffer+h O ) Centrifugation (with filtration) scrap/suspend/centrifuge LC/IC-MS experiment High-Performance Liquid Chromatography (HPLC) metabolism quenching/extraction/lysis (cold methanol; methanol/ammonium bicarbonate; liquid nitrogen into cells/methanol; sonication) adherent mammalian cells suspension mammalian cells suspend/centrifuge LC/IC-MS experiment MECHANISM, METHOD SETUP, EXAMPLES adapted from Junhua Wang, Ph.D. at Thermo 5

6 7/9/18 Retention Mechanisms Retention Mechanisms Phenomenex Phenomenex Mobile Phase Composition Mobile Phase Composition (John Dolan) Phenomenex Phenomenex LC Method Development/Optimization ph Selectivity Column selection Bedding chemistry, dimension, bead size Buffers (mobile phase) Ionic strength, ph, pairing reagent Fine tune the method Temperature Gradient slope Waters Corporation 6

7 7/9/18 ph Selectivity Organic Modifier Selection Waters Corporation Waters Corporation Column Configurations/Applications Column ID (mm) Length Particle Flow Rate Applications Sensitivity Type (mm) Size (mm) Ranges Increase Nano Proteomics, - nl/min Sample Limited 37 PTM Characterization Capillary.3, , Peptide Mapping 1 ml/min LC/MS Micro Bore , High Sensitivity ml/min LC/MS Narrow , Sample Limited. 5 Bore ml/min LC/MS Analytical , 5 1- Analytica; 1 ml/min Semi-prep Small Scale -- ml/min protein purification Preparative , 7-6 CombiChem -- ml/min purification Imtakt Phenomenex Waters Others Column Comparisons by Vendors Details refer to the pdf files LC Tips on Peak Shape Issues LC Tips on Peak Shape Issues Agilent Technologies Agilent Technologies 7

8 7/9/18 LC Tips on Peak Shape Issues Watch for Sample Carry-Over (Contamination) Quantitation Qualitative studies Agilent Technologies Metabolomics in MS&P On-going Projects: Quantification of target metabolite from infected tissue Amino acid analysis in fish embryos Detection/quant. of modified carbohydrate phytochemicals in broccoli extracts Lipidomics of fatty tissue TMAO/TMA analysis from plasma and urine Sample type: Plant extracts Coating material Environmental Bio-fluid Tissues etc. Mass spec analysis UNTARGETED METABOLOMICS : Q -TOF TARGETED METABOLOMICS : QQQ Q EXACTIVE PLUS 1 5 T F T IC R Quadrupole Time-of-Flight (Q-TOF) Benefits: Higher resolution & mass accuracy All ions recorded in parallel TOF ID m/z Oligo Oligo Oligonucleotide Standard BPC x1. Bruker Standards 178 and 6 RPLC with Hexafluoro isopropanol (HFIP) mm & TEA 15 mm. Q1 q Oligo Oligo Oligo Oligo Oligo Oligo Time [min] Olistd_815_3_BE_1_171.d: BPC -All MS Bruker maxis User Manual 8

9 L_6996_61_1_BB5_1_113.d: -MS, 3.7min # m/z L_6996_6191_1_BB5_1_118.d: +MS, 3.7min # m/z L_6996_61_1_BB5_1_1181.d: -MS, 18.min # m/z L_6996_631_1_BB5_1_16.d: +MS, 18.min # m/z 7/9/18 Mass Spectrum at. min Untargeted Metabolomics (RPLC-MS) x1 ID m/z.75 Oligo Oligo Oligo x1 1. Oligo Oligo Oligo x1 Oligo Oligo MS,.min # m/z -MS,.min # m/z m/z -MS,.min # m/z m/z x1 5 6 x MS - MS + L_6996_61_1_BB5_1_113.d: BPC -All MS L_6996_6191_1_BB5_1_118.d: BPC +All MS Time [min] RPLC-MS at 3.7 min Untargeted HILIC-LC-MS x1 5 MS - x1 5 6 MS - L_6996_61_1_BB5_1_1181.d: BPC -All MS x1 5 MS + x MS + L_6996_631_1_BB5_1_16.d: BPC +All MS Time [min] HILIC-MS at 18. min x1 RPLC vs HILIC (Comparison of Elution) MS - Pyro-L-glutamine-L-glutamine x1 1.5 L_6996_6191_1_BB5_1_118.d: EIC 58.11±.1 +All MS RPLC x MS +. x1 5 3 L_6996_631_1_BB5_1_16.d: EIC 58.11±.1 +All MS HILIC Time [min] 9

10 Compound name: Clo Correlation coefficient: r =.9997, r^ = Calibration curve:.679 * x Response type: Internal Std ( Ref ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Include, Weighting: 1/x, Axis trans: None Compound name: Imi Correlation coefficient: r = , r^ = Calibration curve: * x Response type: Internal Std ( Ref ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Include, Weighting: 1/x, Axis trans: None /9/18 Triple Quadrupole MS (QQQ) QQQ Scan Modes for MSMS Analysis Benefits: Simple, ion filter Ideal for quantification Q3 Product Ion Scan Precursor Ion Scan Select Scan Dissociate Dissociate Scan Select Q1 q Neutral Loss Scan Scan Dissociate D Scan Selected Reaction Monitoring (SRM) Select Dissociate Select % Targeted Metabolomics 1181_339_STD_GS_IS L_5 sample after GS, centrifuge, filter, fresh prep in 5%B Imi;6.;393.7; /9. % _339_STD_GS_IS L_5 sample after GS, centrifuge, filter, fresh prep in 5%B Clo;6.1;381.81; /169. % min F:MRM of 1 channel,es+ TIC.6e+6 min F1:MRM of 1 channel,es+ TIC 3.6e+6 Quantitation Using Area Under the Curve (AUC) Std curve QCs Samples Blanks 1181_339_STD_GS_IS L_5 sample after GS, centrifuge, filter, fresh prep in 5%B 1 6./13. min F3:MRM of 1 channel,es+ TIC IS;6.5;379.75;155.17e+7 % min Response ng/ml Response ng/ml Thank you! Yu Cao Arpad Somogyi Vicki Wysocki Entire CCIC MSP Facility 1