Peptide Quantitation Ning Tang, Ph. D. With An Agilent 6410 QQQ … · Peptide MRM Page 1 June...

Peptide MRM

June 26th, 2008Page 1

Peptide Quantitation With An Agilent 6410

QQQ System

Ning Tang, Ph. D.Application scientistLife Sciences and Chemical AnalysisAgilent Technologies

Peptide MRM

June 26, 2008Page 2

Agenda

• Introduction

• HPLC-Chip/QQQ system

• Reproducibility and sensitivity

• Case studies

• Conclusions

Peptide MRM

June 26, 2008Page 3

Agilent Proteomics Biomarker Workflow

Candidates

Proteolytic Digest

Extraction

Depletion

Fractionation

Extraction

Identification

6520 QTOF

6410 QQQ

Candidate Biomarker

Identification

Validation

SAMPLE1SAMPLE2

DATA

Peptide MRM

June 26, 2008Page 4

Jump From Discovery Phase to Validation Phase

• Reduce the time needed for analysis• Increase throughput• Improve CV• Reduce cost

Research Clinical

# of samples <100

Hundreds -

thousands

Time Months -years

minutes -hours

# of proteins 50-500 1-20

Cost $100k-1M $10-100

CV 20-50% 3-5%

Peptide MRM

June 26, 2008Page 5

Agilent HPLC-Chip/QQQ LCMS TechnologyNanospray chip configuration brings new era in high sensitivity quantitation

NanoLC system foranalytical chromatography

HPLC Chip Cube system

QQQ LCMS

CapLC pump for sampleloading on enrichment column

Sensitivity: down to low amolDynamic range: up to 105

Peptide MRM

June 26, 2008Page 6

HPLC-Chip/MS Interface:Fluid Connections to the HPLC-Chip

Rotor

Stator

inner rotor

outer rotor

Stator

RotorSide View

Nanopump

AutosamplerWaste

Microvalve

HPLC-Chip

Peptide MRM

June 26, 2008Page 7

Chromatographic PerformanceProtein Digest Mixture

Reduced MS complexity + reduced ionization competition = improved ID

0

1

2

3

0

1

2

3

12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5Time [min] 10 12 14 16 18 20 22 Time [min]

0

2

4

6

x107

Intensity

0.0

0.5

1.0

1.5

2.0

x108

EIC m/z 985.615.7 secs

6 secs

32% increase

Conventional nanospray LC/MS

Conventional nanospray LC/MS

HPLC-Chip/MS HPLC-Chip/MS

EIC m/z 985.6

Peptide MRM

June 26, 2008Page 8

Retention Time Reproducibility

RT SD %RSDEIC 487.8 3.618 0.014 0.40

EIC 752 3.788 0.011 0.29

EIC 740.6 5.018 0.010 0.20

EIC 874.4 3.968 0.012 0.31

EIC 653.6 4.289 0.012 0.28

EIC 511.7 3.681 0.012 0.31

EIC 722.7 3.547 0.012 0.35

EIC 778 4.143 0.010 0.23

EIC 526.3 4.399 0.015 0.34

EIC 547.5 4.472 0.011 0.25

EIC 746.7 5.196 0.011 0.20

EIC 519.1 4.142 0.011 0.26

EIC 508.2 4.972 0.011 0.23

EIC 582.4 4.679 0.011 0.23

EIC 461.9 3.905 0.012 0.30

EIC 474 4.759 0.011 0.22

EIC 628 4.584 0.010 0.22

Extracted ion chromatograms for 17 peaks from a BSA tryptic digest (50 fmol on-column)

RT reproducibility evaluated using 69 repeat injections

Peptide MRM

June 26, 2008Page 9

Triple Quadrupole Mass SpectrometerExtending Outstanding Performance

6410 Series – Triple Quad High performance Hyperbolic quadrupoles

Chip Cube (Ultimate sensitivity) capability

Extended mass range (2000 amu)

No crosstalk Hexapole Collision Cell

Optimizer – automatic optimization

Scheduled MRMChip system

QQQ MS

Peptide MRM

June 26, 2008Page 10

Agilent’s New Axial Acceleration Collision Cell

Collision Gas

+ Axial Acceleration Potential -

Overcomes memory or cross-talk effects!- using high speed ion transport

Maximum sensitivity- using wide mass range hexapole design

Simple to operate- no complicated wave forms

Beam Turn-off Characteristics

10

100

1000

10000

100000

-500 0 500 1000 1500 2000

microseconds

Arb

. Uni

ts

mz922 mz118

Beam Turn-off Characteristics

10

100

1000

10000

100000

-500 0 500 1000 1500 2000

microseconds

Arb

. Uni

ts

mz922 mz118

600μsec

350μsec

Sub millisecond ion transport

3rd

Quadrupole

1st

Quadrupole

Beam shaper

HEXAPOLE RODS

Entrance lens

Exit lens Beam shaper

Peptide MRM


Parent Ions EnterCollision Cell -

Collide withAr Gas andDissociates

Q1 is Parked AllowingOnly Parent Ions

of a Single m/z to Passto the Collision Cell

Q3 is Parked PassingOnly Product Ions

of a Single m/zto the Detector

Ion GuideTransports

+/- Ions to Q1

+/- Ions andNeutrals Formedin API Source

APISource

Detector

Energy

Triple Quadrupole: SRM

Peptide MRM


Mass Hunter OptimizerCompound Entry & Method Setup

Peptide MRM


Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Compounds (10/block)

Cycle Time (sec)

Max Coincident

Cycle Time (sec)

MRM

50 80

A simple comparison of MRM and Scheduled MRM

0.2 0.4 0.4 0.320 40 40

Scheduled MRM

0.5 0.8 1

30

Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4

0.7100 70

Scheduled MRMIncreased Utility and Performance

2 x shorter cycle times supports narrow chromatographic peaks, more analytes or longer dwell per analyte.

Peptide MRM


Excellent Reproducibility of MS ResponseMRM of HSA Peptide LVNEVTEFAK from 10 amol to 1 pmol (n=6)

All RSDs are within 15%

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.01

0.02

0.05 0.1 0.2 0.5 1 2 5 10 20 50 100

200

500

1000

fmol

RSD

R2 = 0.9975

Peptide MRM


Spike in standard protein digest at different level

Biomarker Discovery and Validation Method Development

Human serum

MARS14 depletion

Denature, reduce, alkylate and digest

OFFGEL fractionation based on peptide pI

Data dependent MS/MS on QTOF

MS profiling on QTOF followed by statistical analysis and

targeted MS/MSQQQ MRM quantitation

Peptide MRM


Limit of Quantitation in the Low Amol RangePeroxidase 10 amol to 10 fmol spiked into 1 µg human serum

2x10

0

0.5

1

1.5

2

+ MRM (480.29999 -> 630.39844) per10a-c2-1ug-r002.d

2x10

0

0.5

1

1.5

2

+ MRM (480.29999 -> 630.39844) per100a-c2-1ug-r001.d

2x10

0

1

2

3

4

+ MRM (480.29999 -> 630.39844) per1f-c2-1ug-r001.d

3x10

0

1

2

3

4

+ MRM (480.29999 -> 630.39844) per10f-c2-1ug-r001.d

Counts vs. Acquisition Time (min)7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

10 fmol peroxidase in 1ug serum

1 fmol peroxidase in 1ug serum

100 amol peroxidase in 1ug serum

10 amol peroxidase in 1ug serum

Peptide MRM


External Quantitation Curve of Peroxidase Peptide DTIVNELR From 10 amol to 10 fmol Spiked into Human Serum

peptide1 - 4 Levels, 4 Levels Used, 12 Points, 12 Points Used, 0 QCs

Concentration (fmol/ uL)-0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5

Res

pons

es 4x10

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

y = 5287.8283 * x + 29.6860R^2 = 0.99866123

peptide1 - 4 Levels, 4 Levels Used, 12 Points, 12 Points Used, 0 QCs

Concentration (fmol/ uL)-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

Res

pons

es 4x10

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

y = 5287.8283 * x + 29.6860R^2 = 0.99866123

100amol

1fmol10amol

R2 = 0.9988

Peptide MRM


Absolute Protein Quantification in the

Context of Non-clinical Drug Safety Evaluation

UCD Conway InstituteUniversity college DublinAndAgilent Technologies

Vehicle Low Dose High Dose

Day 2,4,15 Day 2,4,15 Day 2,4,15 Day -3/-4, 1/2, 3/4, 12/13

HistopathologyTranscriptomics

Proteomics


Proteomics

TranscriptomicsProteomics

MetabonomicsClinical Biochemistry


VehicleVehicle Low DoseLow Dose High DoseHigh Dose

Day 2,4,15 Day 2,4,15 Day 2,4,15 Day -3/-4, 1/2, 3/4, 12/13


Proteomics


Proteomics

TranscriptomicsProteomics



Collins B. C. et al. ASMS 2008 MPQ 477

Peptide MRM


Experimental Design

Rat liver lysate were prepared from rats treated with troglitazone or

vehicle control

Peptides and MRM transitions were selected using Peptide Selector in Spectrum Mill and 13C, 15N labeled

peptides were synthesized

Catalase was selected based on previous 2D-DIGE data

1 mg of soluble protein extract was reduced, alkylated, acetone

precipitated and trypsin digested

The liver digest were spiked with the isotope-labeled peptides and analyzed by Agilent 6410 QQQ system

Peptide MRM


Using Spectrum Mill Peptide Selector for Optimizing MRM Transitions

Chemically reactive residues(Cys = C, Met = M, Trp = W)

Residues with variable PTM

Peptides adjacent to multiple cleavage site

Size of the peptides

Uniqueness of the sequence in the database

Peptide MRM


Peptide Selector – Catalase Results

Peptide MRM


Catalase Peptide EAE – Peptide Selector

5x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

+ Product Ion (5.44-5.70 min, 32 scans) (832.20 -> **) EAE_CE25_Prodion_02.d

1085.68

560.29413.30

201.20 727.49

832.19330.20120.10 616.99

1232.78

Counts vs. Mass-to-Charge (m/ z)100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

Peptide MRM


External Calibration on Catalase PeptideLinearity : five order of magnitude

External quantitation curve of catalase peptide L*AQEDPDYGLR from 78 amol to 7800 fmol

LAQ* - 6 Levels, 5 Levels Used, 12 Points, 10 Points Used, 0 QCs

Concentration (fMol)-500 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 850

Res

pons

es 6x10

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

y = 765.2752 * x + 1585.2251R^2 = 0.99648783

LAQ* - 6 Levels, 5 Levels Used, 12 Points, 10 Points Used, 0 QCs

Concentration (fMol)0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

Res

pons

es 6x10

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

y = 765.2752 * x + 1585.2251R^ 2 = 0.99648783

78aMol

78fMol

780 fMol

R2 = 0.9965

+ MRM (642.5 -> 720.4) SeqA_02-r001.d

Acquisition Time (min)15 15.2 15.4 15.6 15.8 16 16.2 16.4 16.6 16.8

Cou

nts 2x10

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.516.136

Acquisition Time (min)15 15.2 15.4 15.6 15.8 16 16.2 16.4 16.6 16.8

Cou

nts 2x10

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

642.5 -> 720.4 , 642.5 -> 964.5 , 642.5 -> 564.3 , 642.5 -> 320.3

Ratio=32.0Ratio=23.0Ratio=22.0

RSD < 6%

78 amolS/N = 12.0

Peptide MRM


Catalase Quantitation Results

Peptide MRM


Catalase Quantitation Results

Sample PeptideCatalase (fmol/ug protein)

Catalase (pg/ug protein)

Fold Change EAETFPFNPFDLTK

Fold Change LAQEDPDYGLR

Fold Change

2D-DIGE

EAETFPFNPFDLTK 8.84 14.63Vehicle Treated

LAQEDPDYGLR 4.61 5.89

1.00 1.00 1.00

EAETFPFNPFDLTK 32.69 54.36Troglitazone Treated

LAQEDPDYGLR 10.13 13.00

3.7 2.2 1.45

Peptide MRM


Quantitation of Protein Phosphorylation Using

MRM

6x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5+ Scan (5.524-6.123 min, 38 scans) Erk_1ul.d Subtract (1) Deconvoluted (1)

70034.59

69954.86

70111.59

69902.80

70191.7669823.62 70260.88 70343.88

Counts vs. Deconvoluted Mass (amu)69600 69700 69800 69900 70000 70100 70200 70300 70400

+2 PO4

+3 PO4

+4 PO4

Erk1 intact protein

Tang, N. et al. ASMS 2008 TPS 565

Peptide MRM


Selection of MRM Transitions

TY: IADPEHDHTGFLTEYVATRy5

b14 Precursor ion Product ions

545.3 615.3 782.5TY

[M+3H] 3+ y5 b142+

753.3 615.3 979.9t202

[M+2H] 2+ y5 y162+

753.3 979.9 695.3y204

[M+2H] 2+ y162+ y5

780.0 647.6 695.3t202y204

[M+2H] 2+ y163+-H3PO4 y5

t202: IADPEHDHTGFLTEYVATRy16

y5

P

y204: IADPEHDHTGFLTEYVATRy16

y5

P

t202y204: IADPEHDHTGFLTEYVATRy16

y5

P P

Peptide MRM


Chromatographic Separation of the Four Peptide Standards Allowed the Selection of the Same Q1 and Q3 Transitions For Two Different Peptides

4x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

+ MRM (545.29999 -> 615.29688) mix500f-r001.d

Counts vs. Acquisition Time (min)9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.

TY

t202t202y204

y204

Peptide MRM


QC of Incoming Synthetic Peptides

5x10

0.5

1

1.5

+ TIC MRM (** -> **) 1211-r001.d

* 1774762

3x10

0

1

2

3

4

5

+ TIC MRM (** -> **) 1212-r001.d

* 11177

3x10

0

0.5

1

1.5

2

2.5+ TIC MRM (** -> **) 1213-r001.d

* 5914* 7869

3x10

0

0.5

1

1.5

+ TIC MRM (** -> **) 1214-r001.d

* 4029

Counts vs. Acquisition Time (min)12 12.2 12.4 12.6 12.8 13 13.2 13.4 13.6 13.8 14 14.2 14.4 14.6 14.8 15 15.2 15.4 15.6 15.8 16 16.2 16.4 16.6 16.8 17 17.2 17.4 17.6

TY

t202

y204

t202y204

Percentage of componentsPeptides TY t202 y204 t202y204TY 100% - - -

t202 0.6% 99.4% -99.8%

-

-y204 0.2% - -

t202y204 0.4% 2% 97.6%

Peptide MRM


T432t13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs

Concentration (fmol/ ul)-25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525

Rel

ativ

e R

espo

nses 1x10

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

y = 0.1069 * x + 0.2840R^2 = 0.99593214

t202

R2 =0.9959T432t13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs

Concentration (fmol/ ul)-1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5

Rel

ativ

e R

espo

nses

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4 y = 0.1069 * x + 0.2840R^2 = 0.99593214

0.5fm2.5fm

5fm

T432tY434y13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs


Rel

ativ

e R

espo

nses 1x10

-0.20

0.20.40.60.8

11.21.41.61.8

22.22.42.62.8

33.23.43.63.8

4y = 0.0764 * x + 0.2516R^2 = 0.99673108

t202y204

R2 =0.9967T432tY434y13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs


Rel

ativ

e R

espo

nses

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6y = 0.0764 * x + 0.2516R^2 = 0.99673108

0.5fm 2.5fm 5fm

Standard Curves of Four Peptides

TY13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs


Rel

ativ

e R

espo

nses 2x10

-0.1

00.10.20.3

0.40.50.6

0.70.80.9

1

1.11.21.3

1.41.51.61.7

1.81.9

y = 0.3630 * x + 0.0337R^2 = 0.99816859

TY

R2 =0.9981TY13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs


Rel

ativ

e R

espo

nses

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4y = 0.3631 * x + 0.0429R^2 = 0.99829044

0.5fm2.5fm

5fm

Y434y13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs


Rel

ativ

e R

espo

nses 2x10

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.51.6

1.7

1.8

y = 0.3447 * x + 0.1682R^2 = 0.99564716

y204

R2 =0.9956Y434y13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs


Rel

ativ

e R

espo

nses

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5y = 0.3447 * x + 0.1682R^2 = 0.99564716

0.5fm

2.5fm5fm

Peptide MRM


Quantitation of the Degree of Phosphorylation at T202 and Y204 in Active Erk1 Protein

peptide % Molar ratio RSD (n=9)

TY 20% 0.13

t202 25% 0.15

y204 21% 0.12

t202y204 34% 0.08

In this active Erk1 sample, 59% of T202 and 55% of Y204 were phosphorylated

Peptide MRM


HPLC-Chip/QQQ System

Provide high sensitivity and large dynamic range

Robust and stable nanoflow with HPLC-Chip

Good retention time and MS detection reproducibility

Peptide selector helps choosing MRM transitions

Peptide MRM


Acknowledgement

UCD Conway InstituteUniversity college DublinStephen Pennington

Ben Collins

Thomas Lau

William M. Gallagher

NovonordiskAlbrecht Gruhler

Sanofi-AventisJean-Charles Gautier

Agilent TechnologiesChristine Miller

Peter Stone

Peptide MRM


Slide 34

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Peptide Quantitation Ning Tang, Ph. D. With An Agilent 6410 QQQ … · Peptide MRM Page 1 June...

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