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April 11th, 2018

7th Czech Mass Spectrometry Conference

Next Generation Technology for Reproducible and Precise Proteome ProfilingLars Kristensen, Ph.D.Application and training specialist

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Agenda

1) Introduction and Q Exactive HF-X

2) High Resolution DIA Workflow

3) DDA+ Workflow (Label free quantification)

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Sample Preparation Separation Data Acquisition Data Analysis

Large Scale Proteomics

The Goal: Standardized, High Throughput Proteomics

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• Unrivaled precision in precursor quantitation• Maximize complete, reproducible quantitation

across samples• Minimize ‘missing values’ among samples

Unsurpassed quantitative precision and reproducibility

Cellular signaling studiesMechanism of action studiesPTM profiling

Unparalleled proteome coverage and dynamic range

• Highest depth of proteome coverage and quantitative insight

• Robust quantitative precision

Biospecimen profilingDigital archiving

High-Resolution DIA Workflow DDA+ Workflow

Flexible Quantitative Workflow Solutions

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HyperQuad Mass Filter with Advanced Quadrupole Technology

Ultra-High Field Orbitrap

Mass Analyzer

HCD Cell

C-Trap

High Capacity Transfer Tube

ElectrodynamicIon Funnel

Advanced Active Beam Guide

Q Exactive HF-X – New architecture

Brighter ion beam:2-3x More sensitive Dedicated transient for TMT 10plex: 50 K res

Optimized Scan Matrix with accelerated HCD:

40 Hz MS/MS

Improved S/N for intact proteins: 7.5 K res

Advanced DDA for bottom-up and top-down:

Advanced Peak Determination

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Q Exactive HF-X – Basis for increased speed and sensitivity

32 msmaxIT

32 ms

MS/MSQ Exactive HF

Q Exactive HF-X

18-22 Hz• Brighter ion beam (ion funnel),

reduced scan overhead, and accelerated HCD (aHCD) is boosting acquisition speed

• Advantage for both MS and MS/MS mode

• Fast and high quality MS/MS acquisition up the 40 Hz with new 16 msec transient (7,500 resolution setting)

Longer fill timeReduced scan overheadaHCD

maxIT32 ms32 ms

28 Hz

Comparable fill timeReduced scan overheadaHDC

16 ms 16 ms

40 Hz

Orbitrap detection

Maximum fill time with precursor ions

Inter and intra scan overheads

maxIT

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High-Resolution DIA Workflow

STANDARDIZEDQUANTITATIVE PRECISE REPRODUCIBLE

Sample Preparation Separation Data AnalysisData Acquisition

High-Resolution DIA Workflow

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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 Pulsar™ software

Designed for Speed and Coverage

• 150 µm ID x 150 mm, • Sensitivity and robustness

using Cap flow (≈ 1.2 µL/min)• 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

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Designed for high throughput DIA data analysis

Key Benefits

• Spectronaut Pulsar™ is specifically developed for the analysis of DIA & SWATH data sets

• Data analysis with retention time correction based on spiked reference peptides using iRT Kit

• Spectral library generation from MaxQuant, Protein Pilot, and Thermo Scientific™ Proteome Discoverer™

• Direct visualization of qualitative and quantitative results on protein level including FDR and P-values

• Processing of very large experiments (1000s of runs)

• Fast data analysis speed in less than 2 min per run

• DirectDIA™ library generation from DIA data (no need to create a DDA-based library)

Spectronaut Pulsar Software

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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• More robust• Less protein and peptide id’s

050

100150200250300350400

Nano Capillary Capillary

Gradient (min)

Total analysis time (min)

Tim

e (m

in)

# of

IDs

02000400060008000

10000

Nano Capillary Capillary

Total Proteins

0

20000

40000

60000

80000

Nano Capillary Capillary

Total Peptides

# of

IDs

for Triplicates

60 m

in

60 m

in

30 m

in

60 m

in

60 m

in

30 m

in

60 m

in

60 m

in

30 m

in

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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 tot

al

%CV

Quantitation variance

Proteins

Peptides

0

10000

20000

30000

40000

50000 Peptides

90%

74%

0

1000

2000

3000

4000

5000 Proteins

83%68%

Complete QuantificationAND CV<20%AND CV<10%

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High-Resolution DDA+ Workflow

STANDARDIZEDQUANTITATIVE PRECISE REPRODUCIBLE

Sample Preparation Separation Data AnalysisData Acquisition

DDA+ Workflow

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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

using Cap flow (≈ 1.2 µL/min)• RT stability <1% observed for

350 injections

• UltiMate 3000 RSLCnanosystem

• Direct inject or pre-concentration mode

• Viper fittings

• Increased acquisition speed • Advanced precursor determination• Same # of protein IDs half the time

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Key Benefits

• Enables large scale, multiplex proteomic studies (TMT 11-plex) 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

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time

Prot

ein

ID

Q Exactive HF-X MS Q Exactive HF MS

time

Pept

ide

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• Same # of protein ID in half the time

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

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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

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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

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0%

20%

40%

60%

80%

100%

0 10 20 30 40 50

% o

f pep

tides

%CV

Peptide quantitation variance

81%

97%

22525 peptidescompletely quantified

DDA+ Enable Unrivaled Quantitative Precision

0%

20%

40%

60%

80%

100%

0 10 20 30 40 50

% o

f pro

tein

s

%CV

Protein quantitation variance

89%98%

3329 proteins completely quantified

Quantitation Precision StandardizationReproducibility

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DDA+ Workflow: Greater Reproducibility Between Samples

0500

1000150020002500300035004000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Prot

ein

grou

ps

Cumulative replicatesDDA+ Traditional DDA

Proteins93%

62%

0

5000

10000

15000

20000

25000

30000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Pept

ide

grou

ps

Cumulative replicatesDDA+ Traditional DDA

83%

Peptides

20%

Quantitation Precision StandardizationReproducibility

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0

500

1000

1500

2000

2500

3000

3500

4000

4500

Germany 1 Germany 2 USA 1Av

erag

e #

of P

rote

in G

roup

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

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Questions?