HPLC-MS/MS and Metabolomics Yu Cao Ph.D. CCIC MS&P Summer Workshop August 17, 2015 Publication in Metabolomics 8000 6000 Number of publications per year Search engine: Web of Science Topic: metabolomics 4000 2000 0 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 1
Applications of metabolomics in cancer research Kathleen A. Vermeersch and Mark P. Styczynski J. Carcinogenesis, 2013, 12, doi: 10.4103/1477-3163.113622 Metabolomics short course at ASMS 2014 2
INNOVATION Metabolomics: the apogee of the omics trilogy Gary J. Patti, Oscar Yanes and Gary Siuzdak Molecular Cell Biology, 2012, 13, 263-269 Sample preparation 3
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/MS 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 4
Types of Separation Technologies for Molecules 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 2 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 5
Types of Separation Technologies for Molecules Method Separation based on Separation done using Further steps Liquid-liquid Extraction Partitioning in one of two liquid phases Glass ware Solid-phase Extraction Adsorption/ partitioning onto solid sorbent Cartridges, disks, filters, plates Solid-Phase Extraction Uses chromatographic particles Packed-bed column cartridges or similar Well established commercial technology (1978) 1000s literature refs Clean extracts Good recovery for polar analytes Sample must be in liquid state Driving force: gravity, pressure, vacuum Automation cartridges 96 well plate disk http://solutions.3m.com/wps/portal/3m/en_us/empore/extraction/ 6
Solid-Phase Extraction Types of Chromatography Normal Phase Non-polar mobile phase Polar stationary phase Manufacturer Brand Name Reversed Phase Most common Polar mobile phase Non-polar stationary phase Waters Varian SEP-PAK OASIS BondElute Ion Exchange Buffer/Ionic mobile phase Baker BakerBond Cationic/Anionic exchange stationary phase 3M Empore Supelco Supelclean + Many Others 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 7
(KNO 3 ) n K + pure analyte (control) engine oil contaminated parking lot oil same as b) after organic matter removal by SPE Gapeev, A. and Yinon, J. J. Forensic Sci. 2004, 49 Types of Separation Technologies for Molecules Method Liquid-liquid Extraction Solid-phase Extraction Separation based on Partitioning in one of two liquid phases Adsorption/ partitioning onto solid sorbent Separation done using Glass ware Cartriges, disks, filters, plates Further steps Dialysis/Ultrafiltration Molecular weight/size SlideAlyzer/tubing 8
Dialysis Tubing or Slide A-Lyzer Diff MWCO ranges 0.1 0.5 ml capacity Useful for biologicals Sample loading here Spin filters polyethersulfone membrane (Vivaspin, ex) volumes from 100 μl to 20 ml, with a range of molecular weight cutoff values from Mr = 3 000-100 000 Types of Separation Technologies for Molecules Method Liquid-liquid Extraction Solid-phase Extraction Separation based on Partitioning in one of two liquid phases Adsorption/ partitioning onto solid sorbent Separation done using Glass ware Cartriges, disks, filters, plates Further steps Dialysis/Ultrafiltration Molecular weight/size SlideAlyzer, tubing, spin filter Precipitation Solubility 9
HPLC Mechanism, Method Setup, Examples Retention Mechanisms Phenomenex 10
Retention Mechanisms Phenomenex Mobile Phase Composition Phenomenex 11
Phenomenex Phenomenex 12
Generic LC Method Development Column selection Bedding chemistry, dimension, bead size Buffers (mobile phase) Ionic strength, ph, pairing reagent Fine tune the method Temperature Gradient slope ph Selectivity Waters Corporation 13
ph Selectivity Waters Corporation Organic Modifier Selection Waters Corporation 14
Chart of Mobile Phase Selection Waters Corporation Column Type Column Configurations and Applications ID (mm) Length (mm) Particle Size (m) Flow Rate Ranges Applications Sensitivity Increase Nano 0.1-0.075 150 3.5 100-600 nl/min Capillary 0.3, 0.5 35-250 3.5, 5 1-10 L/min Micro Bore 1.0 30-150 3.5, 5 30-60 L/min Narrow Bore 2.1 15-150 3.5, 5 0.1-0.3 ml/min Analytical 4.6 15-250 3.5, 5 1-4 ml/min Semi-prep 9.4 50-250 5 4-10 ml/min Preparative 21.2 50-250 5, 7 20-60 ml/min Proteomics, Sample Limited PTM Characterization Peptide Mapping LC/MS High Sensitivity LC/MS Sample Limited. LC/MS 2000-3700 100 20 5 Analytica; 1 Small Scale protein purification CombiChem purification -- -- 15
Column Comparison by Vendors Imtakt Phenomenex Waters Others Details refer to the pdf files http://www.imtaktusa.com/products/ http://phx.phenomenex.com/lib/po26681014_w.pdf http://www.waters.com/webassets/cms/library/docs/ 720002241en.pdf LC Tips on Peak Shape Issues 16
LC Tips on Peak Shape Issues LC Tips on Peak Shape Issues 17
Metabolomics in MS&P Projects going on Sample type: Eerie lake project Amino acid analysis in animal models Oligonucleotide project Unknown identification in smoker plasma Honey bee project Drug stability TMAO analysis for breast cancer study Plant extract Coating material Environmental Bio-fluid Tissues etc. Mass spec analysis Untargeted metabolomics: Q-TOF Targeted metabolomics: Q-TOF and QQQ 18
Q-TOF Q1 q2 TOF Benefits: Higher resolution & mass accuracy All ions recorded in parallel Ref: Chemushevich, 2001 Chromatogram of Oligonucleotides std on Q-TOF Intens. x10 4 4 2.0 1.5 3 6 8 1.0 1 5 7 0.5 2 0 5 10 15 20 25 30 Time [min] Olistd_040815_03_BE4_01_171.d: BPC -All MS Sample: Bruker standard 217028 and 206200 (MW: 1K~10K Da) 19
MS of Peak 1 at 2.4 min Intens. x10 4 0.75 0.50 585.6075 1172.2196 MS, 2.4min #145 0.25 0.00 x10 4 1.00 0.75 0.50 0.25 0.00 x10 4 0.8 0.6 0.4 0.2 0.0 402.9755 1300.1652 400 600 800 1000 1200 1400 m/z MS, 2.4min #145 1172.2196 1173.2211 1174.2237 1172.7220 1175.2245 1171 1172 1173 1174 1175 1176 1177 m/z MS, 2.4min #145 585.6075 586.1094 586.6119 587.1097 585.0 585.5 586.0 586.5 587.0 587.5 588.0 m/z MS of Peak 6 at 19.0 min Intens. x10 4 0.75 1669.2783 MS, 19.0min #1138 0.50 1112.5167 0.25 402.9783 0.00 x10 4 1.00 0.75 667.1091 555.7557 834.1345 1668.7765 981.9848 1669.2783 1669.7776 1395.7077 500 750 1000 1250 1500 1750 2000 2250 2500 2750 m/z MS, 19.0min #1138 0.50 0.25 0.00 4000 1670.2807 1670.7858 1671.2796 1671.7823 1668.0 1668.5 1669.0 1669.5 1670.0 1670.5 1671.0 1671.5 1672.0 1672.5 m/z MS, 19.0min #1138 1112.5167 1112.8509 1112.1838 1113.1849 2000 1113.5211 1112.6720 1113.8554 1114.1830 0 1111.5 1112.0 1112.5 1113.0 1113.5 1114.0 1114.5 1115.0 1115.5 m/z 20
Untargeted metabolomics RPLC-MS Intens. x10 5 6 4 MS - L_6996_062014_01_BB5_01_1132.d: BPC -All MS 2 Intens. 0 x10 6 0.8 0.6 0.4 0.2 MS + L_6996_061914_01_BB5_01_1108.d: BPC +All MS 0.0 0 10 20 30 40 50 Time [min] RPLC-MS at 32.7 min Intens. x10 5 1.0 0.8 0.6 540.3352 L_6996_062014_01_BB5_01_1132.d: MS, 32.7min #1963 MS - 0.4 0.2 0.0 Intens. x10 5 452.2813 311.1698 200 400 600 800 1000 L_6996_061914_01_BB5_01_1108.d: 1200 +MS, 1400 32.7min #1965 m/z 496.3465 6 4 MS + 2 0 454.2978 620.4440 664.4708 708.4972 991.6849 200 400 600 800 1000 1200 1400 m/z 21
Untargeted HILIC-LC-MS Intens. x10 5 6 4 MS - L_6996_062214_01_BB5_01_1181.d: BPC -All MS 2 Intens. 0 x10 6 0.8 0.6 0.4 0.2 MS + L_6996_062314_01_BB5_01_1206.d: BPC +All MS 0.0 0 5 10 15 20 25 30 35 Time [min] HILIC-MS at 18.2 min Intens. x10 4 2.0 1.5 1.0 242.0782 302.0988 L_6996_062214_01_BB5_01_1181.d: MS, 18.2min #1092 MS - 0.5 133.0133 611.1400 0.0 Intens. x10 5 200 400 600 800 1000 L_6996_062314_01_BB5_01_1206.d: 1200 +MS, 1400 18.2min #1093 m/z 258.1098 3 MS + 2 1 198.1228 515.2127 0 200 400 600 800 1000 1200 1400 m/z 22
Chromatogram of m/z at 258.1098 RPLC v.s. HILIC Intens. x10 4 L_6996_061914_01_BB5_01_1108.d: EIC 258.1100±0.1 +All MS 1.5 1.0 0.5 Intens. 0.0 x10 5 L_6996_062314_01_BB5_01_1206.d: EIC 258.1100±0.1 +All MS 3 2 1 0 0 10 20 30 40 50 Time [min] Targeted metabolomics Cl N HN N N 100 % 256.0/209.0 Imi;6.24;439232.47;4237493 4.446e+006 O - N + O N Cl N N + O O - Imidacloprid NH N H Clothianidin HN N D D N N + O O - S N D D Cl Imidacloprid-d4 (Int. Std.) 0 12182014_30349_STD_GS_IS L_50 sample after GS, centrifuge, filter, fresh prep in 50%B 100 % 0 12182014_30349_STD_GS_IS L_50 sample after GS, centrifuge, filter, fresh prep in 50%B 100 % 0 5.84 250.0/169.0 260.0/213.0 Clo;6.14;382144.81;3449917 IS;6.25;2232479.75;21550402 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 min F1:MRM of 1 channel,es+ TIC 3.462e+006 min F3:MRM of 1 channel,es+ TIC 2.174e+007 What are we detecting and which instrument is used??? min 23
QQQ Q3 Q1 q2 Benefits: Simple, ion filter Good for quantification MS/MS Scan Modes Product Ion Scan Select Dissociate Scan Precursor Ion Scan Scan Dissociate Select Neutral Loss Scan Scan Dissociate Scan Selected Reaction Monitoring (SRM) Select Dissociate Select 24
Selected Reaction Monitoring Source Q1 (gas) Q3 Detector Select one m/z (fixed V ac /V dc ) MS/MS at different collision energies Thermo provided data base 25
Quantitation upon AUC Compound name: Clo Correlation coefficient: r = 0.999227, r^2 = 0.998455 Calibration curve: 0.67092 * x + 0.0383766 Response type: Internal Std ( Ref 4 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Include, Weighting: 1/x, Axis trans: None Std curve QCs Samples Blanks Compound name: Imi Correlation coefficient: r = 0.998517, r^2 = 0.997036 Calibration curve: 0.730897 * x + 0.110503 Response type: Internal Std ( Ref 4 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Include, Weighting: 1/x, Axis trans: None 340 320 300 280 260 240 220 200 360 340 320 300 280 260 240 220 Response 180 160 140 120 100 80 60 40 Response 200 180 160 140 120 100 80 60 40 20-0 ng/ml -0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 20-0 ng/ml -0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 Bioinformatics in Metabolomics 26
ESI-MS n Libraries # Spectra # Compounds Notes METLIN > 68,000 13,048 Public, no download mzcloud > 416,000 2,975 Public, no download MoNA > 236,000 69,946 Public, no download MassBank > 28,100 > 43,000 Public, downloadable HMDB 9,500 Public, downloadable GNPS > 9,000 ~2200 NP Public, downloadable ReSpect (MS n ) > 9,000 3,595 Public, downloadable NIST14 MS/MS > 234,000 9,344 Commercial Wiley MSforID > 10,000 > 1,200 Commercial MetabolomicsWorkBench Public, no download LipidBlast 200,000 Public, downloadable Lipid Maps 37,500 Public, downloadable LipidSearch >1500,000 lipid ions Commercial Database search Progenesis QI http://www.nonlinear.com/progenesis/qi/how-it-wor 27
Import data Data alignment MS + MS - 28
Peak picking MS + MS - Review deconvolution 29
Identify compounds Review compounds 30
Pathway analysis Other Computational Tools for Metabolomics MS-DIAL Data dependent and/or independent MS/MS experiments MS-FINDER GC/MS data alignment, database search (commercial) 31
Anal. Chem. 2014, 86, 9583-9589 Anal. Chem. 2014, 86, 9358-9361 32
Thank you! 33