The world leader in serving science
2017 Metabolomics Seminars
Pushing the Leading Edge in Protein Quantitation:Integrated, Precise, and Reproducible Protein Quantitation Workflow Solutions
2
3
4
Goal: To detect cancer at an early stage while providing additional therapies to more patients.
Cancer Moonshot
5
“…It is the proteins that comprise most of the biomarkers that are measured to detect cancers,
constitute the antigens that drive immune response and inter and intracellular communications,
and it is the proteins that are the drug targets for nearly every targeted therapy that is being
evaluated in cancer trials today.”
Conrads et al. 2016 Clinical Cancer Research
Proteins Are the Machinery, Markers, and Targets for Cancer
6
Sample Preparation Separation Data Acquisition Data Analysis
Large Scale Proteomics
The Goal: Standardized, High Throughput Proteomics
7
Quantitative
Reproducible
Standardized Methods
Curated Methods
Sound Statistical Analysis
Precise
Desirable
Method• HR DIA
• DDA+
A new standard in quantitative,
sensitivity, accuracy and
precision
Our Solution
Workflows
Versatile Workflow Solutions For Your Needs
8
• Unrivaled precision in precursor quantitation
• Maximize complete, reproducible quantitation
across samples
• Minimize ‘missing values’ among samples
Unsurpassed quantitative precision and
reproducibility
Cellular signaling studies
Mechanism of action studies
PTM profiling
Unparalleled proteome coverage and dynamic
range
• Highest depth of proteome coverage and
quantitative insight
• Robust quantitative precision
Biospecimen profiling
Digital archiving
High-Resolution DIA Workflow DDA+ Workflow
Flexible Quantitative Workflow Solutions
9
High-Resolution DIA Workflow
STANDARDIZEDQUANTITATIVE PRECISE REPRODUCIBLE
Sample Preparation Separation Data AnalysisData Acquisition
High-Resolution DIA Workflow
10
High-Resolution DIA: Unparalleled Proteome Coverage and Dynamic Range
Thermo Scientific™ Q Exactive™ HF-X
Hybrid Quadrupole-Orbitrap MS
Thermo Scientific™
EASY-Spray™ LC Column
Spectronaut™
software
Designed for Speed and Coverage
• 150 µm ID x 150 mm,
• Sensitivity and robustness
• RT stability <1% observed
for 350 injections
• Thermo Scientific™ UltiMate™
3000 RSLCnano system
• Direct inject or pre-
concentration mode
• Thermo Scientific™ Viper™
fittings
Workflow
Thermo Scientific™
UHPLC Systems
• Increased acquisition speed
• Advanced precursor determination
• Same # of protein IDs half the time
11
Designed for high throughput DIA data
analysis
Key Benefits
• Spectronaut™ software is specifically developed
for the analysis of DIA & SWATH data sets
• Data analysis with retention time correction based
on spiked reference peptides-HRM calibration kit
or iRT Kit
• Spectral library generation from MaxQuant and
Thermo Scientific™ Proteome Discoverer™
software search results
• Direct visualization of qualitative and quantitative
results on protein level
• Fast data analysis speed in less than 2 min per
run
Spectronaut Software
12
Balancing Efficiency Without Sacrificing Performance
Nanoflow
• Greater # of proteins
• Greater # of peptides
• Greater sensitivity
• Longer total run times
CapLC
• Greater Efficiency
• Shorter total run time ( 2X)
• Greater throughput
• Comparable protein and peptide id’s
050
100150200250300350400
Nano Capillary Capillary
Gradient (min)
Total analysis time (min)
Tim
e (
min
)
# o
f ID
s
0
2000
4000
6000
8000
10000
Nano Capillary Capillary
Total Proteins
0
20000
40000
60000
80000
Nano Capillary Capillary
Total Peptides
# o
f ID
s
for Triplicates
13
High-Resolution DIA Workflow: Highly Precise Proteome Quantitation
• Maximize depth of coverage
• Robust quantitative precision
• Confident in IDs
• Short analysis time
CapLC DIA, 4ug HeLa, 60min, 120K -> Spectronaut Analysis
0%10%20%30%40%50%60%70%80%90%
100%
0 10 20 30 40 50
% o
f to
tal
%CV
Quantitation variance
Proteins
Peptides
0
10000
20000
30000
40000
50000 Peptides
90%
74%
0
1000
2000
3000
4000
5000Proteins
Complete Quantification
AND CV<20%
AND CV<10%
14
High-Resolution DDA+ Workflow
STANDARDIZEDQUANTITATIVE PRECISE REPRODUCIBLE
Sample Preparation Separation Data AnalysisData Acquisition
DDA+ Workflow
15
DDA+ Workflow: Quantitative Precision and Reproducibility
Q Exactive HF-X MSEASY-Spray LC Column Thermo Scientific™ Proteome
Discoverer™ 2.2 software
Designed for Precision and Reproducibility
Workflow
UHPLC Systems
• 150 µm ID x 150 mm,
• Sensitivity and robustness
• RT stability <1% observed
for 350 injections
• UltiMate 3000 RSLCnano
system
• Direct inject or pre-
concentration mode
• Viper fittings
• Increased acquisition speed
• Advanced precursor determination
• Same # of protein IDs half the time
16
Key Benefits
• Enables large scale, multiplex proteomic studies
and captures confident protein results which
enables confident reproducibility
• Improved Label-free Quantitation
• Feature mapping
• Retention time alignment
• Feature linking across files
• Minora Feature Detector node
• Detects chromatographic peaks and
features according to the specified
quantification approach
• Minimizes ‘missing data points’ and maximizes
quantitative insights
Most comprehensive data analysis
platform for qualitative and
quantitative proteomics research
Proteome Discoverer 2.2 Software
17
time
Pro
tein
ID
Q Exactive HF-X MS Q Exactive HF MS
time
Pe
pti
de
ID
Q Exactive HF-X MS
Q Exactive HF MS
50%
40
%
Maximizing protein identifications
• Quick screening of complex samples
• Quality control of complex samples
• Assessment of sample concentration
Maximizing peptide identifications
• Highest peptide coverage
• Deep proteome analysis
• Spectral library building
Saves time and
samples
in large-scale
proteomics efforts
Maximizing Efficiency for Large Scale Proteomics
18
Maintaining Sensitivity at Increased Robustness – capLC with Q Exactive HF-X MS
• Increased sensitivity on QE HF-X
• Increased peptide identifications at higher robustness
• Higher reproducible protein identifications with reduced total run times
0.0E+00
5.0E+08
1.0E+09
1.5E+09
2.0E+09
2.5E+09
3.0E+09
3.5E+09
4.0E+09
0 10 20 30 40 50 60
Blue= 1 µg (nanoLC) cell lysate digestRed = 4 µg (capLC) cell lysate digest
75 µm ID x 15 cm vs 150 µm ID x 15 cm
Robustness
0.0E+00
5.0E+08
1.0E+09
1.5E+09
2.0E+09
2.5E+09
3.0E+09
0 10 20 30 40 50 60
Q- Exactive HF-X MS
Q- Exactive HF MS
1 ug HeLa + 150 uM x 15 cm Easy Spray
Sensitivity
19
Real Benefit of Using DDA+ Workflow
capLC DDA+, 4ug HeLa, 60min, 120K/7.5K, 19ms, Top 40 -> Proteome Discoverer 2.2
Label-free Quant
DDA+ workflow compared to DDA
• 15% gain in completely quantified
proteins
• 47% gain in completely quantified
peptides
• Maximizes quantitation
Quantified Peptides
Quantified Proteins
97%
50%
97%
82%
New DDA+ Traditional DDA
20
DDA+ Workflow: Protein Quantitation
Quantitation Precision StandardizationReproducibility
Completely quantified proteins
Rep 1
Rep 2
Rep 3
Missing data = sparse quantitationComplete quantitation
82% quantified during MS
97% quantified
from MS and Protein
Discoverer 2.2 software
Rep 1
Rep 2
Rep 3
capLC DDA+, 4ug HeLa, 60min, 120K/7.5K, 19ms, Top 40 -> PD 2.2 Label-free Quant
21
DDA+ Workflow: Near Complete Peptide Quantification
Missing data = sparse quantitationComplete quantitation
50% quantified during MS
97% quantified
from MS and PD 2.2
Completely quantified peptides
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
capLC DDA+, 4ug HeLa, 60min, 120K/7.5K, 19ms, Top 40 -> PD 2.2 Label-free Quant
Quantitation Precision StandardizationReproducibility
22
0%
20%
40%
60%
80%
100%
0 10 20 30 40 50
% o
f p
ep
tid
es
%CV
Peptide quantitation variance
81%
97%
22525 peptides
completely quantified
DDA+ Enable Unrivaled Quantitative Precision
0%
20%
40%
60%
80%
100%
0 10 20 30 40 50
% o
f p
rote
ins
%CV
Protein quantitation variance
89%98%
3329 proteins
completely quantified
Quantitation Precision StandardizationReproducibility
23
DDA+ Workflow: Greater Reproducibility Between Samples
0
500
1000
1500
2000
2500
3000
3500
4000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Pro
tein
gro
up
s
Cumulative replicatesDDA+ Traditional DDA
Proteins
93%
62%
0
5000
10000
15000
20000
25000
30000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Pe
pti
de
gro
up
s
Cumulative replicatesDDA+ Traditional DDA
83%
Peptides
20%
Quantitation Precision StandardizationReproducibility
24
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Germany 1 Germany 2 USA 1A
vera
ge #
of
Pro
tein
Gro
up
s
# of Protein Groups
Location
Instrument Standardization Test
• Three Locations
• HeLa digest metrics
• Protein
• Peptide
• PSMs
• MS/MS
• 60 min gradient
Inter-Site Consistency Across Different Instruments
Quantitation Precision StandardizationReproducibility
25
Objective: Determine analytical robustness and
reliability between laboratories
• Comparability of measurements between laboratories and define
critical parameters
• Ring trial participants adapt a system suitability test protocol to
maintain analytical performance
• Determine the range of accuracy and precision that users can
expect to achieve following the standardized product/assay
• The standardization enables the transfer of measurements
between laboratories
Phase 1
Labs identified, resources secured,
SOP established
Phase 2
Study design, study setup, data
collection
Phase 3
Data review, report-out
Thermo
Fisher
BRIMS
Center
Multi-Center Study to Demonstrate Large-Scale Capabilities
26
The world leader in serving science
28
Questions?
29
optional slides follow
30
Increased Throughput: Cap LC DIA – Q Exactive HF-X MS vs. Q Exactive HX MS
7.0% 8.9 % 6.9 %
QE HF 60mins QE HF-X 60min QE HF-X 30min
* 4ug HELA digest, 3 technical replicates, 120k @ MS1
9.58.0
9.09.8
8.39.59.2
7.98.9
QE HF-X 60mins QE HF 60mins QE HF-X 30mins
Peptide precursors per Protein Group
ID CV% <20% CV% < 10%
46217
37367
42206
34603
28231 2871625990
21095 19772
QE HF-X 60mins QE HF 60mins QE HF-X 30mins
Peptide Precursors(1% FDR)
ID CV% <20% CV% < 10%
Med
ian
CV
%
> 20% Increase
Half the time
Quantitation Precision StandardizationReproducibility
31
• Proteomics is being used to discover and establish the protein landscape of cancer cells or tissues
• Proteomic measurements complement genomics/transcriptomic measurements by reducing the vast number of potentially actionable somatic mutations and identifying genomic variances that might be actionable
Proteomics Complements Genomics
The Age of Multi-Omics Is Here. Are We Ready?
