Research to Routine Workflows for Large and Small Molecules using the Q Exactive HR/MS

Transcription

1 Research to Routine Workflows for Large and Small Molecules using the Q Exactive HR/MS Sept. 22, 2011 Patrick Bennett Director Pharma Strategic Marketing Thermo Fisher Scientific

2 Agenda Definitions Research to Routine Small Vs. Large Molecule Physicochemical Types of instruments/technology and experiments Small molecule regulations vs Large molecule regulations Application of HR/MS Why HR/MS Q Exactive Proven Proteomic workflow tools applied to routine quantitation 2

3 Definitions Pharma traditional pharmaceutical company focused primarily on small molecule drug discovery, development and sales. May have large molecule components and includes CRO s. BioPharma can be same company as Pharma or a company that specifically focuses on biologics based therapeutic agents. Includes CRO s. Biologics complex, natural proteins, blood products, clotting factors, antibodies, mab, peptides, analogues, truncated forms, fusion proteins, non-protein macromolecules RNA/DNA, antisense RNA, aptamer s/oligonucleotides Large molecule biologics; based on molecular weight Small molecule traditional drugs; based on molecular weight. 3

4 4 RESEARCH TO ROUTINE

5 Biologics Development: Research to Routine Unregulated Range of traditional Proteomics experiments through to high throughput screening. Research focus, minor component interest, many analytes, same type of test. Internal based procedures rather than regulated SOP s Regulated Routine and Semi-Routine experiments supporting safety and efficacy studies. Few drugs, many samples, high expense GLP & GMP environment strict SOP s Research Discovery DMPK QC/QA Activities Traditional Proteomics, Protein-protein interactions Pathway analysis Activity, PD Biomarker dev. PTM Analysis Disulfide Mapping Protein Characterization Structural Analysis Immunogenicity Neutralization PK/PD, LBA s, Cell based assays, PTM Optimization, Protein Characterization, Safety, Efficacy, Metabolism, DDI PK/PD, CQA/QbD, Production Characterizations, Drug monitoring 5

6 Small vs Large Chemical Characteristics Characteristic Small Large Molecular Weight <800 >5000 Endogenous No Often Yes Solubility Hydrophobic Hydrophilic Purity Homogeneous Heterogeneous Immunogenic No Yes Conjugated Yes No Valence Monovalent Divalent Kinetics Fast Slow Detection Isotopic Activity/functional Stability Chemical/enzymatic Immunologic/enzymatic Metabolic interference Yes/knowledge Unknown Protein Binding Yes No 6

7 Small vs Large Analytical Methodology Characteristic Small Large Basis of Measurement Analyte Antigen-Ab reaction Detection Direct Indirect Reagents Common and available Unique, not commercial or available Analytes Small Small and macromolecule Sample Preparation Yes No Calibration Curves Linear Non-Linear Assay Environment Organic Aqueous Development Time Weeks Months Technology LC/MS (Affinity based Extraction) LBA: ELISA, RIA, ECL, Multiplexing Stability Drug Drug + Reagents Regulations Very defined and rigid Some flexibility 7

8 8 APPLICATION OF HR/MS

9 Specificity = Resolution + Mass Accuracy Resolution: 10k, 30k, 50k, 100k Butyl-Phthalate, (ubiquitous background ion) 54 ppm apart Relative Abundance Ethinyl-Estradiol, m/z 9

10 High Resolution Ensures Accurate Target Selection R: 35K R: 70K R: 140K ppm m/z Accurate quantification is only possible when the target can be correctly identified. An interference which is 30 ppm apart from the target can be completely separated using high-resolution of 140,000 using the Q Exactive. 10

11 When does HR/MS make sense? Research phase Early to late discovery phases Pre-clinical phase QA/QC - characterization Ligand binding assay development Specificity testing Stability testing Assay troubleshooting, unexpected results Reagents unavailable, difficult to produce, expensive Bridging methods, reagents Biomarker research, multiple biomarkers 11

12 Research to Routine: Range of Experiments for LC/MS Qualitative Identification Confirmation Research Low throughput Discovery Development Medium throughput Verification Quantitative Routine High throughput Optimized assays Traditional Proteomics, Metabolomics, Metabolism, Biomarker Research Translational Research, Biopharma, Metabolomics, Metabolism Drug Discovery, Various Biomarker Pharma & Biopharma Quantitation, Leachables, Extractables, Impurities, QA/QC Orbitraps Q Exactive Triples All Q TOF All Q TOF Triples & Q Trap 12

13 Q Exactive TM Hardware Innovations S-Lens ion source Quadrupole mass filter Advanced signal processing 13

14 Specifications/Details Thermo Scientific HyperQuad mass filter Mass range: m/z Linear range: 4-5 orders of magnitude Variable precursor isolation width selection from 0.4 Da to full mass range Resolution : up to 140,000 17k, 35k, 70k, 140k at m/z 200 scan speed dependent on resolution setting Sources: ESI probe compatible with liquid flow rates of < 1 μl/min to 1 ml/min without splitting APCI source compatible with liquid flow rates of 50 μl/min to 2 ml/min without splitting Nanospray/microspray 14

15 What Do We Gain by Selected Ion Monitoring? In Full MS, total C-trap charge capacity is shared between multiple signals of different intensity Signal-to-noise ratio becomes dependent on the ratio of compound of interest to other analytes-much less so in SIM! In Orbitrap instruments, SIM could become MRM without any additional overhead! N= N= Full MS S/N = 745 IT= ms SIM (10amu) For the same target: S/N = 5400 IT= ms NL: 1.94E8 [ ] Lowest signal NL: 1.12E8 [ ] Lowest signal Gain in sensitivity (7x) 6000 Sensitivity gain 5 10 x with SIM mode The gain will be higher in more complex matrices S/N (spectrum) Caffeine S/N (FMS) S/N (SIM10)

16 16 QUANTITATION

17 Traditional Workflow (Triple Quadrupoles) NCEs Tune MRM PP Sample Clean Up Bioanalysis PK Estimates (NCE only) LC-SRM Analysis Tuning MRM takes time & requires some level of expertise MRM methods are not easily transferable between platforms from different vendors, which makes scalability difficult. Peptides require determination of charge state and the optimal SRM transition, which is again platform dependent. Expertise required, even for a basic assay. Limits the number of transitions (duty cycle & no. of scans per analyte). Difficult to automate set-up to get sequence information expertise required. 17

18 HRMS Workflow (Q Exactive) NCEs PP Sample Clean Up Bioanalysis PK Estimates (NCE) PK Estimates Metabolites LC-HRMS No compound dependent tuning required easier to use/faster to set-up Post-acquisition data analysis Providing PK data as well as critical new information (metabolites, biomarkers) More value in terms of the Fail Fast paradigm Multiple Analytes (IS) Peptides Post-Acquisition Data Query 18

19 Quantitation Summary of Small Molecules on Q-Exactive Q Exactive Full Scan Compound LOQ Curve Fit Oxycodone 50 fg L - 1/X2 Paroxetine 100 fg Q - 1/X2 Ketoconazole 50 fg L - 1/X2 Clonazepam 500 fg Q - 1/X2 Verapamil 50 fg Q - 1/X2 Clopidogrel 50 fg L - 1/X2 Q Exactive Targeted-SIM LOQ Curve Fit 50 fg L - 1/X2 50 fg L - 1/X2 50 fg L - 1/X2 500 fg Q - 1/X2 50 fg L - 1/X2 50 fg L - 1/X2 Triple Quad Vantage (SRM) LOQ Curve Fit 50 fg L - 1/X2 50 fg L - 1/X fg Q - 1/X2 100 fg L - 1/X2 10 fg Q - 1/X2 50 fg Q - 1/X2 Q-Exactive achieves similar quantitation performance of above compound mixture as Vantage 19

20 Uroguanylin in Glucagon Peptide Matrix (Full Scan) Q Exactive Test-003 # RT: AV: 6 NL: 1.17E6 T: FTMS + p ESI Full ms [ ] Relative Abundance [M+2H] [M+H] m/z 20

21 Uroguanylin Singly Charged Species Test-003 # RT: AV: 6 NL: 1.32E5 T: FTMS + p ESI Full ms [ ] R=25584 z= R=25350 z=1 Uroguanylin was quantified by summing the first 3 isotopes of both singly and doubly charged ions Relative Abundance R=25986 z= R=26333 z= R=26133 z= R=25043 z= R=26044 z= m/z 21

22 Uroguanylin Doubly Charged Species Test-003 # RT: AV: 6 NL: 1.13E5 T: FTMS + p ESI Full ms [ ] R=36727 z= R=35417 z= Relative Abundance R=36214 z= R=36408 z= R=35710 z= R=32095 z= R=33958 z= m/z 22

23 Q Exactive: 100 pg/ml to 10,000 pg/ml Uroguanylin in Glucagon Peptide Matrix (t-sim) R 2 =0.9977, Linear 1/x Uroguanylin Y = *X R^2 = W: 1/X^2 Linear Dynamic Range 1 x 10(4) Area Area Uroguanylin Y = *X R^2 = W: 1/X^ ng/ml ng/ml

24 Insulin Quantitation Results - Plasma Nominal Concentration (ng/ml) Replicate # Mean Calculated Concentration Stdev % CV

25 Exendin Quantitation Results - Plasma Nominal Concentration (ng/ml) Replicate # Mean Calculated Concentration Stdev % CV

26 GLP-1 Quantitation Results - Plasma Nominal Concentration (ng/ml) Replicate # Mean Calculated Concentration Stdev % CV

27 PROTEOMICS RESEARCH TO ROUTINE 27

28 Workflow Steps Define the Experiment Identify the protein sequence under study Selection of Peptides Literature search, in silico digestion, BLAST searchers to ensure sequence specificity WRT to the background matrix Selection of Targeted m/z values Sensitivity based on intense fragmentation, selectivity WRT background Verification of Targeted m/z values Comparison of experimental LC-MS and MS/MS parameters to references (incorporation of recombinant protein analysis and/or heavy labeled synthetic peptides Optimization of Targeted m/z values Keeping only the best peptides/product ions, collision energies, scheduled time windows 28

29 Workflow for Targeted Protein Quantitation Development Sample Preparation Mass Spectrometry Data Processing MS Heavy Labeled Protein Protein(s) Digest 1. Ionization (electrospray) 2. MS (full spectra) m/z Quantification Heavy Labeled Peptides PRTC Kit 3. MS Pre-cursor selection 4. Fragmentation (CID, HCD, ETD) Separation HPLC 5. MSn (Product Ion spectra) Reference Database Intensity [counts] MS/MS y?? y?? Peptide sequence verification Time [M+2H]²?-H?O, [M+2H]²?-NH? y?²? y?? y?²?-h?o, y?²?-nh? y?? b?? b?? y??-nh? y?? b?? y?? m/z Domon and Aebersold Nature Biotechnology 2010, 7(28),

30 Target Verification Using Heavy Labeled Peptides Relative Abundance Relative Abundance NL: 1.71E NL: 5.33E LC VFQSWWDR m/z VFQSWWDR m/z Time (min) Relative Abundance MS VFQSWWDR MS/MS VFQSWWDR VFQSWWDR VFQSWWDR m/z

31 31 HeavyPeptide TM AQUA TM Standards

32 HeavyPeptide TM AQUA TM Benefits HeavyPeptide AQUA products enable absolute quantification of all proteins in a sample. The HeavyPeptide AQUA kits can now be prepared with covalent modifications, such as phosphorylation, which are chemically identical to naturally occurring post-translational modifications (PTMs). As a result, the HeavyPeptide AQUA kits are an extremely cost-effective solution, enabling researchers to identify and quantify peptides of interest much faster, with significantly increased precision. This answers the need for relative and absolute quantification of the expression levels for all proteins in complex samples. This is essential since PTMs significantly increase the size of proteomes over their corresponding genomes. The HeavyPeptide AQUA product range produces a clear and consistent gain in efficiency, transparency and reproducibility of experiments. 32

33 Extracted from: C:\Scott Peterman\Backup\Marketing_Monthly_Updates\2011_Marketing_Plan\Thermo_Instruments\Seed_Unit_Requests\Amgen\Control_trainer_180grad_inj1.raw #6273 RT: ITMS, CID, z=+2, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da b₃ y₂ y₅ y₆ Extracted from: C:\Scott Peterman\Backup\Marketing_Monthly_Updates\2011_Marketing_Plan\Thermo_Instruments\Seed_Unit_Requests\Amgen\Control_trainer_180grad_inj1.raw #2982 RT: ITMS, CID, z=+1, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da y₈ [M+1H]+-H₂O b₁₀+-h₂o y₁₂² b₁₃+ y₇+ b₉+-h₂o25 y₉ b₁₄+ y₃+-nh₃ b₅+-h₂o b₇+ b₈ b₁₄+-nh₃, y₁₄ y₁₀ y₃+ y₁₁²+-h₂o, y₁₁²+-nh₃ y₁₁+ 20 y₁₂+ b₁₄ Extracted from: C:\Scott Peterman\Backup\Marketing_Monthly_Updates\2011_Marketing_Plan\Thermo_Instruments\Seed_Unit_Requests\Amgen\Control_trainer_180grad_inj1.raw #4001 RT: ITMS, CID, z=+2, Mono m/z= b₉+ Da, MH+= Da, Match Tol.=0.8 Da b₁₃+-h₂o y₆+ m/z b₈ y₇ b₁₁+ b₆+ y₉+ b₁₀ a₇+-nh₃ b₁₂+-h₂o b₅ y₈+ b₈+-h₂o b₉+-h₂o b₁₁+-h₂o b₁₄+-h₂o y₇ y₁₃+ y₁₂+ b₇+-h₂o y₆ [M+2H]²+ y₅ y₇² b₂ m/z a₃+-nh₃ b₃ y₃ b₄ b₅ m/z Experimental Purified Protein Stock Solution Treatment Digestion PRTC Kit Control H 2 O 2 treated (40 hrs) ph 8.0 treated (40 hrs) Intensity [counts] (10^3) Relative Abundance Time (min) Intensity [counts] (10^3) Intensity [counts] (10^3) Sequence coverage, AUC, PTMs, %CVs, RT confirmation Sequence for Spectral Libraries 33

34 Pierce Peptide Retention Time Calibration Kit + Pinpoint 1.2 Software What s New? Peptide Retention Time Calibration Kit Pinpoint 1.2 Software for Targeted Protein Quan Application: Quickly assess and optimize chromatography and MS instrument performance Predict peptide retention times using calculated hydrophobicity factors Predict peptide elution across multiple instrument platforms Improve quantification and increase multiplexing with optimized scheduled SRM windows Incorporation of the PRTC kit provides a system QC, normalization, and RT correlation across experiments and instrumental platforms. Key features High-purity 15 synthetic heavy peptides mixed at equimolar ratio Elutes across entire chromatographic gradient Fully automated using Pinpoint 1.2 QC page Pinpoint 34

35 Peptide Retention Time Calibration (PRTC) Kit (Heavy Peptides) # Sequence Observed Mass ( Z=2) Hydrophobicity Factor (HF) 1 SSAAPPPPPR GISNEGQNASIK HVLTSIGEK DIPVPKPK IGDYAGIK TASEFDSAIAQDK SAAGAFGPELSR ELGQSGVDTYLQTK GLILVGGYGTR GILFVGSGVSGGEEGAR SFANQPLEVVYSK LTILEELR NGFILDGFPR ELASGLSFPVGFK LSSEAPALFQFDLK

36 Retention Time Analysis Comparison with Internal Standards x R 2 = x R 2 =

37 Conclusions Quanfirmation enabled characterization and quantification were performed in 1 experiment Separating quantification (MS-level) and qualitative enables reprocessing data for targeted peptide expansion Introduction of PRTC kit enabled method reproducibility for the AS, LC, and MS methods PRTC kit provides direct relationship of calculated hydrophobicity factors to measured retention times and scalability Absolute and/or relative quantitation using heavy peptides Entire method is integrated with Proteome Discoverer and Pinpoint 37

38 Acknowledgments Kevin Cook Zhiqi Hao Scott Peterman Thank You 38