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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
June 26, 2008Page 11
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
June 26, 2008Page 12
Mass Hunter OptimizerCompound Entry & Method Setup
Peptide MRM
June 26, 2008Page 13
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
June 26, 2008Page 14
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
June 26, 2008Page 15
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
June 26, 2008Page 16
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
June 26, 2008Page 17
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
June 26th, 2008Page 18
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
HistopathologyTranscriptomics
Proteomics
TranscriptomicsProteomics
MetabonomicsClinical Biochemistry
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
HistopathologyTranscriptomics
Proteomics
HistopathologyTranscriptomics
Proteomics
TranscriptomicsProteomics
MetabonomicsClinical Biochemistry
MetabonomicsClinical Biochemistry
Collins B. C. et al. ASMS 2008 MPQ 477
Peptide MRM
June 26, 2008Page 19
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
June 26, 2008Page 20
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
June 26, 2008Page 21
Peptide Selector – Catalase Results
Peptide MRM
June 26, 2008Page 22
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
June 26, 2008Page 23
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
June 26, 2008Page 24
Catalase Quantitation Results
Peptide MRM
June 26, 2008Page 25
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
June 26th, 2008Page 26
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
June 26, 2008Page 27
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
June 26, 2008Page 28
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
June 26, 2008Page 29
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
June 26, 2008Page 30
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
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
-0.20
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
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
-0.4
-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
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 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
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
-0.4
-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
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 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
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
-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
June 26, 2008Page 31
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
June 26, 2008Page 32
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
June 26, 2008Page 33
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
June 26, 2008Page 34
Slide 34
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