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Research to Routine Workflows for Large and Small Molecules using the Q Exactive HR/MS
Sept. 22, 2011
Patrick BennettDirector Pharma Strategic MarketingThermo Fisher Scientific
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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
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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.
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RESEARCH TO ROUTINE
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Biologics Development: Research to Routine
Research Discovery DMPK QC/QA
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
Activ
ities 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
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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
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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
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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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492.2665
492.2495
492.2634
R: 35K R: 140K
m/z
492.2661
492.2511
R: 70K
30ppm
High Resolution Ensures Accurate Target Selection
• Accurate quantification is only possible when the target can becorrectly identified.
• An interference which is 30 ppm apart from the target can becompletely separated using high-resolution of 140,000 using theQ Exactive.
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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
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RoutineHigh throughput
Optimized assays
Pharma &Biopharma
Quantitation, Leachables, Extractables,
Impurities, QA/QC
DevelopmentMedium throughput
Verification
Translational Research,
Biopharma,Metabolomics,
MetabolismDrug Discovery,
Various Biomarker
ResearchLow throughput
Discovery
TraditionalProteomics,
Metabolomics, Metabolism,
Biomarker Research
Research to Routine: Range of Experiments for LC/MS
All Q TOF All Q TOF Triples &Q Trap
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
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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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• 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 chargecapacity is shared between multiplesignals of different intensity
• Signal-to-noise ratio becomesdependent on the ratio of compoundof interest to other analytes-muchless so in SIM!
• In Orbitrap instruments, SIM couldbecome MRM without any additionaloverhead!
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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 MS
SIM (10amu)
S/N = 745IT= 0.245 ms
For the same target:S/N = 5400IT= 1.321 ms
Lowest signal250330
Lowest signal28240
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195.082 195.084 195.086 195.088 195.09 195.092 195.094
S/N
(spe
ctru
m)
S/N (FMS) S/N (SIM10)
Gain in sensitivity (7x)
Caffeine
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QUANTITATION
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Traditional Workflow (Triple Quadrupoles)
NCEs
TuneMRM
PP Sample Clean Up
Bioanalysis
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
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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-HRMSNCEs
PP Sample Clean Up
Bioanalysis
PK Estimates (NCE)
PK Estimates Metabolites
Multiple Analytes (IS)
Peptides
Post-Acquisition Data Query
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Quantitation Summary of Small Molecules on Q-Exactive
Q ExactiveFull Scan
Q ExactiveTargeted-SIM
Triple Quad Vantage (SRM)
Q-Exactive achieves similar quantitation performance of above compound mixture as Vantage
Compound LOQ Curve Fit
Oxycodone 50 fg L - 1/X2Paroxetine 100 fg Q - 1/X2
Ketoconazole 50 fg L - 1/X2Clonazepam 500 fg Q - 1/X2Verapamil 50 fg Q - 1/X2
Clopidogrel 50 fg L - 1/X2
LOQCurve
Fit50 fg L - 1/X250 fg L - 1/X250 fg L - 1/X2
500 fg Q - 1/X2
50 fg L - 1/X2
50 fg L - 1/X2
LOQ Curve Fit
50 fg L - 1/X250 fg L - 1/X2
1000 fg Q - 1/X2100 fg L - 1/X2
10 fg Q - 1/X2
50 fg Q - 1/X2
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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
0
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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Are
a
Linear Dynamic Range 1 x 10(4)
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Insulin Quantitation Results - Plasma
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 5.96
2.5 4 2.81 0.0568 2.025 4 4.79 0.112 2.33
10 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 3.62
1000 4 979 36.7 3.75
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Exendin Quantitation Results - Plasma
Nominal Concentration
(ng/mL)
Replicate #
Mean Calculated
ConcentrationStdev % CV
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 0.40
1000 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 - Plasma
Nominal Concentration
(ng/mL)
Replicate #
Mean Calculated
ConcentrationStdev % CV
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 2.50
2.5 4 2.69 0.0578 2.155 4 5.36 0.126 2.36
10 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 3.35
1000 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
200 300 400 500 600 700 800 900 1000 1100m/z
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Inte
nsity
[cou
nts ]
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
4 6 80
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10001020
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60
7080
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NL: 1.71E5
NL: 5.33E6
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561 562
LC
563 564 565 566 567 568 569 570 571 572 573m/z
VFQSWWDR
VFQSWWDR
Rel
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bund
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567.2720
567.7727
568.2739
568.7753562.2684
562.7704
MS
200 300 400 500 600 700 800 900 1000
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
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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.
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Experimental
Control
H2O2 treated (40 hrs)
pH 8.0 treated (40 hrs)
Purified Protein Stock Solution
Treatment Digestion PRTC Kit
0 10 20 30 40 50 60 70 80 90 100 110120 130Time (min)
020406080100020406080100
Rel
ativ
e Ab
unda
nce
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
400 600 800 1000 1200 1400 1600
m/z
0
20
40
60
80
100
Inte
nsity
[co
un
ts] (1
0^3
)
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
600 800 1000 1200 1400 1600
m/z
0
5
10
15
20
25
30
Inte
nsity
[co
un
ts] (1
0^3
)
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: 41.53 ITMS, CID, z=+1, Mono m/z=1712.74365 Da, MH+=1712.74365 Da, Match Tol.=0.8 Da
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
200 300 400 500 600 700 800 900 1000
m/z
0
10
20
30
40
50
60
70
80
90
Inte
nsi
ty [c
ou
nts
] (1
0^3
)
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: 55.85 ITMS, CID, z=+2, Mono m/z=525.29041 Da, MH+=1049.57353 Da, Match Tol.=0.8 Da
Sequence for Spectral Libraries
Sequence coverage, AUC, PTMs, %CVs, RT confirmation
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• 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
Pierce Peptide Retention Time Calibration Kit + Pinpoint 1.2 Software
Pinpoint
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Peptide Retention Time Calibration (PRTC) Kit (Heavy Peptides)
# Sequence Observed Mass( Z=2)
HydrophobicityFactor (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
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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 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
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Acknowledgments
• Kevin Cook• Zhiqi Hao• Scott Peterman
Thank You