Research to Routine Workflows for Large Molecules using ... · SOP’s Activities Traditional Proteomics, Protein-protein interactions Pathway analysis Activity, PD Biomarker dev

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

European Bioanalytical Forum 16 November 2011

Patrick BennettDirector Pharma Strategic MarketingThermo Fisher Scientific

2

Agenda• 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

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RESEARCH TO ROUTINECONTRAST OF LARGE VS. SMALL

4

Biologics Development: Research to Routine

Unregulated Regulated•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

•Routine and Semi-Routine experiments supporting safety and efficacy studies. •Few drugs, many samples, high expense•GLP & GMP environment – strict SOP’s

Act

iviti

es Traditional Proteomics,Protein-protein interactionsPathway analysisActivity, PD Biomarker dev.

PTM AnalysisDisulfide MappingProtein CharacterizationStructural AnalysisImmunogenicityNeutralization

PK/PD, LBA’s, Cell based assays, PTM Optimization,Protein Characterization, Safety, Efficacy, Metabolism, DDI

PK/PD, CQA/QbD, Production Characterizations,Drug monitoring

5

Small vs Large Chemical Characteristics

Characteristic Small LargeMolecular Weight <800 >5000Endogenous No Often YesSolubility Hydrophobic HydrophilicPurity Homogeneous HeterogeneousImmunogenic No YesConjugated Yes NoValence Monovalent DivalentKinetics Fast SlowDetection Isotopic Activity/functionalStability Chemical/enzymatic Immunologic/enzymaticMetabolic interference Yes/knowledge UnknownProtein Binding Yes No

B. De Silva, Bristol-Myers Squibb

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Small vs Large Analytical Methodology

Characteristic Small LargeBasis of Measurement Analyte Antigen-Ab reactionDetection Direct IndirectReagents Common and available Unique, not commercial

or availableAnalytes Small Small and macromoleculeSample Preparation Yes NoCalibration Curves Linear Non-LinearAssay Environment Organic AqueousDevelopment Time Weeks MonthsTechnology LC/MS (Affinity based

Extraction)LBA: ELISA, RIA, ECL, Multiplexing

Stability Drug Drug + ReagentsRegulations Very defined and rigid Some flexibility

B. De Silva, Bristol-Myers Squibb

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APPLICATION OF HR/MS

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Specificity = Resolution + Mass Accuracy

Resolution: 10k, 30k, 50k, 100k

279.12 279.14 279.16 279.18 279.20m/z

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Ethinyl-Estradiol, 279.17434

Butyl-Phthalate, 279.15909 (ubiquitous background ion)

54 ppmapart

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100 ppb Ethinyl-Estradiol – 100k vs 10 K Res

Ethinyl-Estradiol

17 18 19Time (min)

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16.68 17.81 18.07 18.9417.59 19.04

Res = 10,000Phthalate

E Estradiol

17 18 19Time (min)

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18.03 19.3217.22 18.1716.40 18.76

17.73Res = 100,000

Phthalate

E Estradiol

Both Resolution and Mass Accuracy Essential

10

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

RoutineHigh throughput

Optimized assays

Pharma &Biopharma

Quantitation, Leachables, Extractables,

Impurities, QA/QC

DevelopmentMedium throughput

Verification

Translational Research,Biopharma,

Metabolomics,Metabolism

Drug Discovery,Various Biomarker

ResearchLow throughput

Discovery

TraditionalProteomics,

Metabolomics, Metabolism,

Biomarker Research

All Q TOF All Q TOF Triples &Q Trap

Research to Routine: Range of Experiments for LC/MS

QuantitativeQualitative ▪ Identification ▪ Confirmation ▪

Q ExactiveOrbitraps Triples

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Q Exactive TM Hardware Innovations

• S-Lens ion source• Quadrupole mass filter• Advanced signal processing

13

Specifications/Details

• Thermo Scientific HyperQuad mass filter• Mass range: 50-4000 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

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Scan Speed and Operation Modes

• Compatibility for UHPLC up to 70k resolution• Compatibility for >5 second wide peaks at 140,000

resolution• Full MS with high resolution accurate mass detection • Selected Ion Monitoring (SIM) and Multiplex SIM (mSIM)• MS/MS of isolated ions with high resolution accurate mass

detection• ‘All Ion Fragmentation’ in the HCD collision cell• Source fragmentation of all ions in the source region • Positive/negative ion switching• Data Dependent on-the-fly decision making • Timed SIM for scheduled data

15

• Sensitivity gain 5 – 10 x with SIM mode

• The gain will be higher in more complex matrices

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!

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195.0876N=248402.81

195.0877N=20741.58

NL: 1.94E8[150.00-2000.00]

NL: 1.12E8[190.10-200.10]

Full MSS/N = 745IT= 0.245 ms

Lowest signal250330

SIM (10amu)For the same target:S/N = 5400IT= 1.321 ms

Lowest signal28240

Gain in sensitivity (7x)

Caffeine

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QUANTITATION

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Traditional Workflow (Triple Quadrupoles)

NCEsNCEs

TuneMRMTuneMRM

PP Sample Clean Up

PP Sample Clean Up

BioanalysisBioanalysis

PK Estimates (NCE only)

PK Estimates (NCE only)

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.

LC-SRM Analysis

18

HRMS Workflow (Q Exactive)

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

LC-HRMSNCEsNCEs

PP Sample Clean Up

PP Sample Clean Up

BioanalysisBioanalysis

PK Estimates (NCE)

PK Estimates (NCE)

PK Estimates Metabolites

PK Estimates Metabolites

Multiple Analytes (IS)

Multiple Analytes (IS)

PeptidesPeptides

Post-Acquisition Data Query

Post-Acquisition Data Query

19

Quantitation Summary of Small Molecules on Q-Exactive

LOD LLOQ LOD LLOQ LOD LLOQOxycodone 1 5 5 10 1 5Buprenorphine 50 100 50 50 50 50Paroxetine 1 10 1 5 10 10Ketoconazole 50 100 1 50 1 50Clonazepam 1 10 5 50 10 10Verapamil 1 1 5 5 1 5Alprazolam 1 5 5 10 10 50Reserpine 1 10 10 50 5 10Clopidogrel 1 5 50 50 5 10

CompoundTSQ QE Full Scan (70K) QE tSIM (70K)

All Regression linear 1/x2 or quadratic 1/x2

20

Uroguanylin in Glucagon Peptide Matrix (Full Scan) – Q Exactive

Test-003 #487-492 RT: 2.28-2.30 AV: 6 NL: 1.17E6T: FTMS + p ESI Full ms [820.00-1700.00]

850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700m/z

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871.66876

1161.88527

981.82791

1667.61833

1177.54283

834.31346

939.13037 1026.997631472.23880

891.16079

1128.20213 1195.51589 1689.600491408.69556 1529.258531266.47914 1356.68300 1607.28170

[M+H]+[M+2H]2+

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Uroguanylin Singly Charged SpeciesTest-003 #487-492 RT: 2.28-2.30 AV: 6 NL: 1.32E5T: FTMS + p ESI Full ms [820.00-1700.00]

1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675m/z

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1667.61833R=25584

z=1

1668.62129R=25350

z=1

1669.62036R=25986

z=1

1670.62126R=26333

z=1

1671.62188R=26133

z=11672.62227

R=25043z=1

1673.62198R=26044

z=1

Uroguanylin was quantified by summing the first 3 isotopes of both singly and doubly charged ions.

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Test-003 #487-492 RT: 2.28-2.30 AV: 6 NL: 1.13E5T: FTMS + p ESI Full ms [820.00-1700.00]

833.5 834.0 834.5 835.0 835.5 836.0 836.5 837.0 837.5 838.0m/z

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834.31346R=36727

z=2

834.81445R=35417

z=2

835.31439R=36214

z=2

835.81451R=36408

z=2

836.31443R=35710

z=2836.81542R=32095

z=2

837.40331R=33958

z=4

Uroguanylin Doubly Charged Species

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UroguanylinY = -11732.2+366792*X R^2 = 0.9977 W: 1/X^2

0 100 200 300 400 500 600 700 800 900 1000 1100ng/mL

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20000000

40000000

60000000

80000000

100000000

120000000

140000000

160000000

180000000

200000000

220000000

240000000

260000000

280000000

300000000

320000000

340000000

360000000

380000000

400000000

Are

aQ Exactive: 100 pg/mL to 10,000 pg/mL – Uroguanylin in Glucagon Peptide Matrix (t-SIM) R2=0.9977, Linear 1/x2

UroguanylinY = -11732.2+366792*X R^2 = 0.9977 W: 1/X^2

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0ng/mL

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Area

Linear Dynamic Range 1 x 10(4)

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Insulin Quantitation Results

Nominal Concentration

(ng/mL)Replicate #

Mean Calculated

ConcentrationStdev % CV

0.25 4 0.260 0.0300 11.50.5 4 0.434 0.0501 11.51 4 0.906 0.0540 5.962.5 4 2.81 0.0568 2.025 4 4.79 0.112 2.3310 4 10.3 0.284 2.7725 4 27.9 0.247 0.8950 4 44.0 1.35 3.08100 4 92.8 4.29 4.62250 4 276 9.99 3.621000 4 979 36.7 3.75

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Exendin Quantitation Results

Nominal Concentration

(ng/mL)

Replicate #

Mean Calculated


5 4 5.143 0.418 8.1310 4 9.693 0.520 5.3625 4 24.37 1.22 5.0050 4 45.46 0.705 1.55100 4 92.83 13.0 14.0250 4 261.7 5.31 2.03500 4 507.3 2.03 0.401000 4 966.0 25.2 2.612500 4 2675.6 60.7 2.275000 4 5205 169 3.2410000 4 9740 150 1.54

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GLP-1 Quantitation Results

Nom inal Concentration

(ng/mL)

Replicate #

Mean Calcu lated


0.1 4 0.099 0.0098 9.910.25 4 0.251 0.0205 8.160.5 4 0.534 0.0337 6.311 4 0.905 0.0226 2.502.5 4 2.69 0.0578 2.155 4 5.36 0.126 2.3610 4 9.99 0.120 1.2025 4 26.6 0.664 2.50250 4 50.6 0.562 1.11100 4 80 0.692 0.86250 4 243 14.2 5.86500 4 462 15.5 3.351000 4 860 7.90 0.925000 4 5383 62.8 1.1710000 4 9796 164 1.67

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PROTEOMICS RESEARCH TO ROUTINE

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Define the Experiment

Selection of Peptides

Selection of Targeted m/z values

Verification of Targetedm/z values

Optimization of Targetedm/z values

Identify the protein sequence under study

Literature search, in silico digestion, BLAST searchers to ensure sequencespecificity WRT to the background matrix

Sensitivity based on intense fragmentation, selectivity WRT background

Comparison of experimental LC-MS and MS/MS parameters to references(incorporation of recombinant protein analysis and/or heavy labeled synthetic peptides

Keeping only the best peptides/product ions, collision energies, scheduled time windows

Workflow Steps

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Workflow for Targeted Protein Quantitation Development

Protein(s)

Digest

Separation HPLC

Heavy Labeled Peptides

Heavy Labeled Protein

PRTC Kit

Time →

2. MS(full spectra)

1. Ionization(electrospray)

y??

1024.58

b??

949.42

y??290.27

y??‐NH?

860.53

b??

648.28

b??

247.07

y??

662.37

y?²?

439.35

y?²?‐H?O, y?²?‐NH?

430.38

y??

476.34

[M+2H]²?‐H?O, [M+2H]²?‐NH?553.27

y??

749.47

y??

877.53

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Quantification

Peptide sequenceverification

3. MSPre-cursor selection

4. Fragmentation(CID, HCD, ETD)

5. MSn(Product Ion spectra)

ReferenceDatabase

m/z

MS

Sample Preparation Mass Spectrometry Data Processing

m/z

MS/MS

Domon and Aebersold Nature Biotechnology 2010, 7(28), 710-721

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10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44Time (min)

20.79

20.79

Target Verification Using Heavy Labeled Peptides

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NL: 5.33E6

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561 562

LC

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VFQSWWDR

VFQSWWDR

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567.2720

567.7727

568.2739

568.7753562.2684

562.7704

MS

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877.46

749.45

553.21

476.22430.39

662.33544.29247.12860.61219.15 375.18 512.81 949.36290.14 648.37

759.33 887.38

558.52

486.35444.33

672.40435.22247.09

219.12 517.79 648.31 870.44375.19300.17

VFQSWWDR

VFQSWWDR

MS/MS

VFQSWWDRm/z 567.2720

VFQSWWDRm/z 562.284

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HeavyPeptideTM AQUATM Standards

32

HeavyPeptideTM AQUATM 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.

33

Experimental

Control

H2O2 treated (40 hrs)

pH 8.0 treated (40 hrs)

Purified Protein Stock Solution

Treatment Digestion PRTC Kit

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020406080100

b₁₄⁺‐NH₃, y₁₄⁺1580.73

b₃⁺306.13

b₁₄⁺1597.76

y₃⁺‐NH₃444.35

b₅⁺‐H₂O462.23

y₂⁺333.30

y₃⁺461.28

b₁₃⁺1411.69

y₁₂⁺1438.74

y₁₁²⁺‐H₂O, y₁₁²⁺‐NH₃667.34

y₁₁⁺1351.66

b₈⁺843.46

y₁₂²⁺720.39

y₁₀⁺1264.62

b₇⁺756.43

y₇⁺901.54

y₉⁺1101.60

b₉⁺‐H₂O938.52

b₁₀⁺‐H₂O1039.54

y₈⁺988.54

y₆⁺788.43

y₅⁺687.38

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m/z

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[cou

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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: 88.55 ITMS, CID, z=+2, Mono m/z=872.43262 Da, MH+=1743.85796 Da, Match Tol.=0.8 Da

b₅⁺627.40

a₇⁺‐NH₃782.24

y₆⁺620.40

b₆⁺726.32

b₇⁺‐H₂O809.40

y₁₂⁺1375.65

y₇⁺749.40

b₁₁⁺1237.40

y₁₃⁺1462.69

b₉⁺‐H₂O1075.47

y₈⁺886.46

b₈⁺‐H₂O946.40

b₁₁⁺‐H₂O1219.45

b₈⁺964.44

b₁₄⁺‐H₂O1548.63

b₁₂⁺‐H₂O1320.52

y₉⁺987.59

b₁₀⁺1150.41

b₁₃⁺‐H₂O1419.59

b₉⁺1093.47

b₁₄⁺1566.60

[M+1H]⁺‐H₂O1694.69

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[cou

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y₃⁺376.26

b₅⁺560.39

a₃⁺‐NH₃295.32

[M+2H]²⁺525.22

b₄⁺503.28

b₃⁺340.31

b₂⁺227.16

y₇²⁺412.34

y₅⁺547.22

y₇⁺823.48

y₆⁺710.39

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Sequence for Spectral Libraries

Sequence coverage, AUC, PTMs, %CVs, RT confirmation

34

• 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

Pierce Peptide Retention Time Calibration Kit + Pinpoint 1.2 Software

Pinpoint

35

Peptide Retention Time Calibration (PRTC) Kit (Heavy Peptides)

# Sequence Observed Mass( Z=2)

Hydrophobicity Factor (HF)

1 SSAAPPPPPR 493.7 7.5681

2 GISNEGQNASIK 613.3 15.50003

3 HVLTSIGEK 496.3 15.52207

4 DIPVPKPK 451.3 17.65144

5 IGDYAGIK 422.7 19.15385

6 TASEFDSAIAQDK 695.8 25.8834

7 SAAGAFGPELSR 586.8 25.23967

8 ELGQSGVDTYLQTK 773.9 28.36797

9 GLILVGGYGTR 558.3 32.17702

10 GILFVGSGVSGGEEGAR 801.4 34.51977

11 SFANQPLEVVYSK 745.4 34.96488

12 LTILEELR 498.8 37.30326

13 NGFILDGFPR 573.3 40.41916

14 ELASGLSFPVGFK 680.4 41.18506

15 LSSEAPALFQFDLK 787.4 46.66305

36

Retention Time Analysis – Comparison with Internal Standards

2.14591x + 7.46657 R2 = 0.95902

1.91004x + 11.44465 R2 = 0.97641

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Conclusions

• Quanfirmation enabled characterization and quantification are performed in a single experiment

• Separating quantification (MS-level) and qualitative analysis enables reprocessing data for targeted peptide expansion

• Introduction of PRTC kit enabled method reproducibility for 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

38

Acknowledgments

• Kevin Cook• Scott Peterman, Ph.D.• Zhiqi Hao, Ph.D.

39

THANK YOU

40

Targeted Peptide Verification using HR/AM MS-Level Data

...

844.0 844.4 844.8 845.2 845.6 846.0m/z

05101520253035404550556065707580859095100

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844.4190

844.7531844.0847

845.0871

845.4211

845.7551

Theoretical

844.0 844.4 844.8 845.2 845.6 846.0m/z

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844.4225

844.7565

844.0887

845.0906

845.4250

845.7593

Experimental

4.7 4.1 4.0 4.1Mass Spectral Data XICs

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SMGGKEDLIWELLNQAQEHFGK+3 CS = C112H175N30O35S1

Documents

Research to Routine Workflows for Large Molecules using ... · SOP’s Activities Traditional Proteomics, Protein-protein interactions Pathway analysis Activity, PD Biomarker dev