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Morales
Discovery to Validation: LC/MS Product Specialist
Routine Nano-LC/QQQ Agilent Technologies
for Quantitation of
Peptides
Page 1
Agilent Proteomics Biomarker Workflow
SAMPLE1 DATA
SAMPLE2 Candidate
Extraction Biomarker
Identification
6520
Depletion QTOF Candidates
Fractionation Proteolytic
Digest
Extraction
6410
QQQ
Identification
Validation
Page 2
Biomarker validation workflow
• Perform statistical
analysis for Step 3
identification of
Spectrum Mill
biomarker candidates
• Run samples on Q- • Search QTOF data
TOF for protein ID in using Spectrum Mill
data-dependent
MS/MS mode • Use Spectrum Mill
MRM Builder to
create a list of MRM
Step 1 transitions with RT
Q-TOF
Step 2 and 5
Mass Profiler Pro
• Run samples on
• Integrate the MRM QQQ in Dynamic
chromatograms MRM mode
• Import quantitation
results into MPP to
perform statistical
analysis Step 4
QQQ
Page 3 2/15/2011
Workflow for quantitative peptide analysis using MRM
1. Creation of a (D)MRM acquisiton method from
discovery data
MRM
optimization
QTOF MRM
SpectrumMill MRM Builder
acquisition acquisition
DMRM
Export MRM transitions and optional retention time information acquisition
for optimization or direct (D)MRM acquisition analysis
Verify results from Optimizer for peptides and perform (D)MRM analysis
• Separation Speed
• Peak Capacity
• Reproducibility
Mass Spectrum
• Selectivity
• Acquisition Rate
Page 7 2/15/2011
HPLC-Chip/QQQ LCMS Technology
Nanospray chip configuration brings new era in high sensitivity
quantitation
QQQ LCMS
Page 8
Triple Quadrupole Mass Spectrometer
Extending Outstanding Performance
Page 9
HPLC-Chip/MS Interface:
Fluid Connections to the HPLC-Chip
Autosampler
Waste
Nanopump
Stator
Side View
Rotor
inner rotor
outer rotor
Rotor
Microvalve
Stator
HPLC-Chip
Page 10
Retention Time Reproducibility
RT SD %RSD
EIC 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
Extracted ion chromatograms for 17 peaks from a EIC 778 4.143 0.010 0.23
BSA tryptic digest (50 fmol on-column) 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
5
log individual intensity
4,5
4 Replicate 1
Replicate 2
3,5 Replicate 3
Replicate 4
3 Replicate 5
2,5 N =234
2,5 3 3,5 4 4,5 5 5,5
Peptide
sequence
Precursor
19
Dynamic MRM
Comparison of MRM and Dynamic MRM
Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4
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
MRM
Dynamic MRM
Max Coincident 20 40 40 30
Cycle Time (sec) 0.4 0.4 0.4 0.4
• 2 x shorter cycle times supports narrow chromatographic peaks, more analytes or longer dwell per
analyte.
Page 20
Dynamic MRM – what happens?
Increases
Sensitivity
Improves cycle
time
Provides better
chromatographic
definition
Day 2,4,15 Day 2,4,15 Day 2,4,15 Day -3/-4, 1/2, 3/4, 12/13
Page 23
Experimental Design
Page 24
Using Spectrum Mill Peptide Selector for
Optimising MRM Transitions
Chemically reactive residues
(Cys = C, Met = M, Trp = W)
Uniqueness
Page 25
Spectrum Mill – Peptide Selector
Page 26
Peptide Selector – Catalase Results
Page 27
Catalase Peptide LAQ – Peptide Selector
Page 28
Catalase Peptide EAE – Peptide Selector
Page 29
External Calibration on Catalase Peptides
Linearity : five order of magnitude
780 fMol
0.9965
RSD < 6%
Page 30
Catalase Quantitation Results
Page 31
Quantitation of protein Erk1 protein was quantified
phosphorylation using from depleted human serum.
MRM
+3 PO4
Erk1
+2 PO4 intact
+4 PO4 protein
Peptide MRM
Lorne
Page 32
Selection of MRM transitions
Page 33
Chromatographic Separation of the Four Peptide Standards
allowed the selection of the same Q1 and Q3 transitions for
two different peptides
6 TY
5.5
4.5
4 t202
3.5
t202y204
3
2.5
2 y204
1.5
0.5
0
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 19.5
Counts vs. Acquisition Time (min)
Page 34
TY13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs
Relative Responses T432tY434y13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs
x10 2 y = 0.3630 * x + 0.0337
Relative Responses
x10 1 y = 0.0764 * x + 0.2516
R^2 = 0.99816859 4 R^2 = 0.99673108
1.9
3.8
Relative Responses
1.8 Relative Responses
1.7
1.6
5fm R2 =0.9981 3.6
3.4
5fm R2 =0.9967
3.2
1.5 2.5fm 3
2.5fm
1.4
2.8
0.5fm
1.3 0.5fm 2.6
1.2 2.4
1.1 2.2
1 2
0.9 1.8
0.8 1.6
0.7 1.4
0.6
0.5
0.4
TY 1.2
1
0.8
t202y204
0.3 0.6
0.2 0.4
0.1 0.2
0 0
-0.1 -0.2
-25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 -25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525
Concentration (fmol/ul) Concentration (fmol/ul)
Y434y13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs T432t13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs
Relative Responses
Relative Responses
x10 1 y = 0.1069 * x + 0.2840
R^2 = 0.99564716 R^2 = 0.99593214
1.8 5.5
R2 =0.9956 R2 =0.9959
Relative Responses
Relative Responses
1.7
1.6 5
1.5 5f 4.5 5fm
1.4 2.5f m 2.5fm
1.3 0.5f m 4
1.2
m 3.5
0.5fm
1.1
1 3
0.9
0.8 2.5
0.7
2
0.6
0.5
0.4
0.3
y204 1.5
1 t202
0.2
0.5
0.1
0 0
-0.1
-0.5
-25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 -25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525
Concentration (fmol/ul) Concentration (fmol/ul)
Page 35
Quantitation of the degree of phosphorylation at
T202 and Y204 in active Erk1 protein
% Molar
peptide RSD (n=9)
ratio
TY 20% 0.13
Page 36
From Discovery Mode Peroxidase in Human Plasma
to Validation Step
Page 37
Discovery phase to Validation : MRM Selector
Step-2 Step
Spectrum Mill • Import the MRM list Mass Prof
• Run samples on Q- into QQQ Acquisition
TOF for protein ID in • Search QTOF data software • Integrate the MRM
data-dependent using Spectrum Mill • Run samples on QQQ chromatograms
MS/MS mode. • Use Spectrum Mill in Dynamic MRM • Import quantitation
MRM Selector to mode results into MPP to
create a list of MRM perform statistical
transitions with RT analysis
Step-1 Step-3
Q-TOF QQQ
Page 38
Depleted Human Plasma Sample analysis
Data Dependent
Protein IDs from
Spectrum Mill
Page 39
MRM Selector
Generates MRM method from discovery QTOF data
Page 40
Dynamic MRM
Page 41
Dynamic MRM
Overlaid 2000 MRM chromatograms acquired in a single run using Dynamic MRM
Page 42
Sensitivity :
Peroxidase in plasma matrix (per 0.5ug)
M A B
Page 43
Mass Profiler Professional
Statistical Processing of MRM Data
B1 B2 B3 A1 A2 A3 M1 M2
All Samples
Page 44
Principle Component Analysis
Matrix and 2 different peroxidase levels
Samples at
different
peroxidase
concentrations
were correctly
grouped
together.
Page 45
Hierarchical Clustering
comparing different peroxidase concns.
Page 46