Quantitative Labeling Using Tandem Mass...

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Rosa Viner Omics marketing group September 13, 2018

Quantitative Labeling Using Tandem Mass Tags


Introduction: TMT based relative quantification

3

Problem: Quantitative information about expression level of a protein is essential to understanding its biological role in response to change or disease.

Add another dimension to any experiment by determining the relative abundance of each identified protein

Moving Beyond Qualitative Proteomics

Isobaric Labeling/Tandem Mass Tags™ (TMT)*

* Ting, L. et al. 2011. Nature Methods 8: 937-940 Tandem Mass Tag and TMT are trademarks of Proteome Sciences plc.

HCD fragmentation LC-MS2

One signal

TMTsixplex™

4

How Does Isobaric Mass Tagging ( TMT&iTRAQ) Work?

julian.saba

Text Box

Tandem Mass Tag and TMT are trademarks of Proteome Sciences plc.

5

TMT Labels are Indistinguishable

6

The Difference Only Appears in MSn

7

Synchronous Precursor Selection (SPS) for Accurate Quantification

• Available on the Thermo Scientific™ Orbitrap Fusion™ MS and Thermo Scientific™ Orbitrap Fusion™ Lumos ™ MS

• Select multiple MS2 precursors using a single fill and notched isolation waveform

• Improves the ratio accuracy and at the same time dramatically boosts sensitivity

8

Multinotch MS3 Quantification is Accurate and Sensitive

0

2

4

6

8

10

12

14

126 127N 127C 128N 128C 129N 129C 130N

Yeast

Human

Human 10 : 5 : 2 : 1 : 1 : 2 : 5 : 10 Yeast 1 : 1 : 1 : 1

TMT MS3 Peptides-Quan template in Method Editor *

9

TMT11plex

126.1277261

127.1310809 127.1247610

128.1344357 128.1281158

129.1377905 129.1314706

130.1411453 130.1348254

131.1381802 N NNH

O

OOO

O

N NNH

O

OOO

O

N NNH

O

OOO

O

N NNH

O

OOO

O

N NNH

O

OOO

O

N NNH

O

OOO

O

N NNH

O

OOO

O

N NNH

O

OOO

O

N NNH

O

OOO

O

N NNH

O

OOO

O

TMT10-127N

TMT10-128N

TMT10-129N

TMT10-130N

TMT10-127C

TMT10-128C

TMT10-129C

TMT10-130C

TMT10-131

TMT10-126

**

* **

*

*

*

**

*

**

*

**

*

*

**

***

**

* ****

* ***

*

**

**

**

**

*

*

***

*

*

N NNH

O

OOO

OTMT11-131C

*

*

*

** 131.144999

Reporter Ion Mass

• TMT11-131C can be used in combination with TMT10plex reagents to multiplex 11 different samples for MS analysis

• 11plex data analysis is supported by Proteome Discoverer 2.1-2.3

10

High Resolving Power is Required for Accurate Quantification of the TMT11 plex

QE classic, QE plus Transient 128 ms RP 35K@m/z 200

D20 Orbitraps Transient 96 ms RP 45-50K@m/z 200

D20 Orbitraps Transient 128 ms RP 60K@m/z 200

11

TMT Multiplexing Workflow for Precise Data in Less Time

Unique workflow with potential for massive throughput – improvements in instrument technology

Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ MS with Method Templates

Thermo Scientific™ TMT™ 11-Plex Reagents

Sample Labeling and Preparation

LC-MS/MS (SPS MS3) Analysis

Data Analysis

Thermo Scientific™ Proteome Discoverer™ Software and ProteinCenter™ Software

Current Technology 3500 proteins/2 hours

Next Generation Technology 5000 proteins/2 hours

42,000 252,000 420,000

60,000 300,000 600,000 1.08 million

Advances in Tribrid MS technology

# of

exp

erim

ents

/ 24

h

TMT 6 TMT 10 Label free

# of

exp

erim

ents

/ 24

h

TMT 6 TMT 10 Label free ‘TMT 10’

Number of protein quantifications per day

Isolate proteins

Treat, digest

TMT10 - 126

TMT10 – 127C

TMT10 – 127N

TMT10 – 128N

TMT10 – 128C

TMT10 – 129N

TMT10 – 129C

TMT10 – 130N

TMT10 – 130C

TMT10 – 131

1

2

3

4

5

6

7

8

9

10

Label Mix

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TMT11 – 131C

Select for MS2 Identification Quantitation


TMT QC assay - TMT 11 yeast triple knock out standard

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TMT Standard for TMT Workflow QC and Method Development

• MS and LC method optimization

• QC of mass spec and LC

• PD analysis optimization

“TMT standard will not solve the interference problem, it can accurately and sensitively measure its effects”

J.Paulo et al, JASMAS, 2016, 1620-1625

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TMT QC Assay Standard

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Difference between Proteomics TKO Standard and Pierce TMT TKO standard • TKO standard (Paulo et al, 2016)

• Paulo’s TKO standard uses met6(protein rank19), pfk2(114) and ura2(244) yeast strains. • Paulo et al uses TMT9 to label peptides from those three strains.

Strain#1 Strain#2 Strain#3 Strain#1 Strain#2 Strain#3 Strain#1 Strain#2 Strain#3

met6Δ pfk2Δ ura2Δ

TKO standard

• Pierce TMT11 TKO Standard • Pierce TMT11 standard uses met6,his4(213),ura2(244), and BY4742 parental yeast strains. • The standard uses BY4742 parental strain labeled with 131N and 131C as control channels.

F1, 126 F1, 127N F1, 127C F1, 128N F1, 128C F1, 129N F1, 129C F1, 130N F1, 130C F1, 131N F1, 131CQuan Channels

0

20

40

60

80

100

120

140

160

Ab

un

da

nce

[a

.u.]

100100

121120123125127127

111010

P05694: 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase [OS=Saccharomyces cerevisiae S288C]


0

20

40

60

80

100

120

140

160

180

200

Ab

un

da

nce

[a

.u.]

96104

159155155

131516

146147147

P00815: histidine biosynthesis trifunctional protein [OS=Saccharomyces cerevisiae S288C]


0

20

40

60

80

100

120

140

160

Ab

un

da

nce

[a

.u.]

99101

161614

102100100

113113113

P07259: Protein URA2 [OS=Saccharomyces cerevisiae S288C]

Met6 protein abundance profile met6Δ his4Δ ura2Δ BY4742

TMT11 standard

His4 protein abundance profile Ura2 protein abundance profile


Experimental Set up

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• Take vial out of the freezer and bring to the room temperature, 15 min • Add 40 ul of 0.1%TFA/5% AcN in Water, 500 ng/ul • Or add 40 ul of 5% DMSO/1% FA in Water, 500 ng/ul • Incubate at RT for 15 min and transfer to autosampler vial • Don’t keep more than 1 week at 4 C

TMT11 TKO Sample Reconstitution

18

LC Method: EASY-Spray™ C18 50cm column & UltiMate™ 3000 RSLCnano UHPLC

50 min gradient 120 min gradient

Solvent A: 0.1% formic acid

Solvent B: 100 % Acetonitrile, 0,1% formic acid

Flow rate: 300 nL/min

Injection volume: 1-2 µL

Direct injection

19

LC Method: EASY-Spray™ C18 50cm column & UltiMate™ 3000 RSLCnano UHPLC

50 min gradient 120 min gradient





Trap loading

Time(min) Flow, ul/min

0 20

100, 185 20

Loading pump conditions

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LC Method using EASY-Spray™ C18 50cm column & EASY-nLC™ 1200 HPLC

Time (min)

Flow(nL/min) %B

0 300 5

5 300 10

55 300 40 60 300 90 70 300 90

Time (min) Flow(nL/min) %B

0 300 5 5 300 8

125 300 40 130 300 90 140 300 90





50 min 120 min

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LC Method using EASY-Spray™ C18 50cm column & EASY-nLC™ 1000 HPLC

Time (min)

Flow(nL/min) %B

0 300 5

5 300 7

55 300 32 60 300 90 70 300 90

Time (min) Flow(nL/min) %B

0 300 5 5 300 7

125 300 32 130 300 90 140 300 90




Injection volume: 1 µL

50 min 120 min

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Instrument Method Settings: QExactive Classic and QExactive Plus

Properties QE classic 50 min QE classic 120 min QE + 50 min QE + 120 min

Resolution Full MS 70000 70000 70000 70000

AGC target Full MS 3e6 3e6 3e6 3e6

MS max IT, ms 50 50 50 50

Scan range, m/z 350-1500 350-1500 350-1500 350-1500

Loop count 15 15 15 15

MS2 resolution 35000 35000 35000 35000

MS2 AGC target 1e5 1e5 1e5 1e5

MS2 max IT, ms 120 ms 250 ms 100 ms 120 ms

Isolation Window , Th 1.2 1.2 0.7 0.7

NCE, % 32-34 32-34 32-34 32-34

Intensity threshold 1e4 1e4 1e4 1e4

Peptide match preferred preferred preferred preferred

Dynamic exclusion, s 20 s 45 s 20 s 30 s

First mass, m/z 110 110 110 110

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Instrument Method Settings: QExactive HF and QExactive HF X

Properties QE HF 50 min QE HF 120 min QE HF X 50 min QE HF X 120 min

Resolution Full MS 120000 120,000 120000 120000


MS max IT, ms 50 50 50 50

Scan range, m/z 350-1500 350-1500 350-1500 350-1500

Loop count 20 15 20 15

MS2 resolution 60000( 2.9-45000) 60000(2.9- 45000) 45000 45000

MS2 target 1e5 1e5 1e5 1e5

MS2 max IT, ms 96 120 86 96

Isolation Window, Th 0.4 m/z 0.7 m/z 0.7 m/z 0.7 m/z

NCE, % 32-34 32-34 32-34 32-34

Intensity threshold 1e4 1e4 1e4 1e4

Peptide match preferred preferred preferred , single charge preferred , single charge

Dynamic exclusion, s 20 30 20 30

First mass, m/z 110 110 110 110

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Instrument Method Settings: Fusion, Tune3.1 Templates are Available in Method Editor Properties Fusion SPS 50 min Fusion SPS 120 min Fusion MS2 50 min Fusion MS2 120 min



MS max IT, ms 50 50 50 50

Scan range, m/z 375-1500 375-1500 375-1500 375-1500

Top Speed, s 2 3 2 3

MS2 max IT, ms 50 50 105 120

MS2 Isolation window, Th 1.2(2)-0.7(3)-0.5 (4+) 1.2(2)-0.7(3)-0.5 (4+) 1.2(2)-0.7(3)-0.5 (4+) 0.7(2-3)-0.5 (4+)

MS2 NCE, % 35 35 38-40 38-40

MS2 Intensity threshold 5e3 5e3 5e4 5e4

Dynamic exclusion, s 45, single charge 60, single charge 45, single charge 60, single charge

MS2 Resolution turbo turbo 50000 50000


MS3 AGC target 1e5 1e5

SPS Isolation window, Th 1.3(2)-0.7(3)-0.5 (4+) 1.3(2)-0.7(3)-0.5 (4+)

SPS NCE, % 65 65

SPS max IT, ms 105 120

SPS settings: # notches, mass range

5-10-10

m/z 110-500

5-10-10

m/z 110-500

m/z 110

m/z 110

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Instrument Method Settings: Lumos, Tune3.1 Templates are Available in Method Editor Properties Fusion SPS 50 min Fusion SPS 120 min Fusion MS2 50 min Fusion MS2 120 min



MS max IT, ms 50 50 50 50

Scan range, m/z 375-1500 375-1500 375-1500 375-1500

Top Speed, s 2 3 2 3

MS2 max IT, ms 50 50 86 96

MS2 Isolation window, Th 1.2(2)-0.7(3)-0.5 (4+) 1.2(2)-0.7(3)-0.5 (4+) 1.2(2)-0.7(3)-0.5 (4+) 0.7(2-3)-0.5 (4+)

MS2 NCE, % 35 35 38-40 38-40

MS2 Intensity threshold 5e3 5e3 5e4 5e4

Dynamic exclusion, s 45, single charge 60, single charge 45, single charge 60, single charge

MS2 Resolution turbo turbo 50000 50000


MS3 AGC target 1e5 1e5

SPS Isolation window, Th 1.3(2)-0.7(3)-0.5 (4+) 1.3(2)-0.7(3)-0.5 (4+)

SPS NCE, % 65 65

SPS max IT, ms 86 105

SPS settings: # notches, mass range

5-10-10

m/z 110-500

5-10-10

m/z 110-500

m/z 110

m/z 110


PD 2.2 workflow set up

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I. Quantification Method Add Lot Specific Correction Factors

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2

3

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1. Select “Maintain Quantification Methods” 2. Create new method using TMT 11plex template 3. Add Correction factors from Product data sheet 4. Save new method as TMT11TKOlotXXX standard

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II. Study Set up

1. Select New Study 2. Create Study Name 3. Select Study Directory 4. Select Processing workflow 5. Select Consensus workflow 6. Select Quan.method and control channel 7. Add files

1 2 3

4

5

7

6

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II. Study Set up

1. Select categorical factor 2. Create study factors 3. Set study factors, controls for each of the quan.channels per file

1

2

3

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III. Search Parameters For Processing Workflow

Use 0.02 Da tolerance for MS2 methods and 1.2 Da for SPS methods

Download SwissProt yeast database, taxonomy ID 4932

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III. Consensus Workflow

75 for MS3

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III. Consensus Workflow: New In PD 2.3 SPS Mass Matches

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SPS Mass Matches

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III. Ratios Set up per Individual File

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III. Ratios Set up for Multiple files


Results and Quality Control of LC-MS System

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Search and Quan Results: 1012 Protein Groups, 5777 Unique Peptides, 6372 PSMs

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Result Statistics

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Pathway Analysis: Δmet6 & Δura2

• Reactome

• Pyrimidine biosynthesis

• Metabolism of amino acids and derivatives

• Nucleobase biosynthesis

• KEGG

• Biosynthesis of antibiotics

• Alanine, aspartate and glutamate metabolism

• Pyrimidine metabolism

• Glycine, serine and threonine metabolism

• Biosynthesis of secondary metabolites

• One carbon pool by folate

• Glyoxylate and dicarboxylate metabolism

• BioCyc

• UMP biosynthesis II

• urea cycle

• Nitrogen Compounds Metabolism

• Pyrimidine Ribonucleotides De Novo Biosynthesis

• de novo biosynthesis of pyrimidine ribonucleotides

Δura2 Over-represented Pathways:

Comparison: Δura2 and Δmet6

https://apps.thermofisher.com/apps/lsms/oc Diagram created with Omics Comparator

https://apps.thermofisher.com/apps/lsms/oc

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Detailed Result for Selected Protein- KO Protein Met6

Scaled Abundances

Median Ratios

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Detailed Result for Selected Peptide Group from KO Protein Met6

Peptide Abundances

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182; 3.15%

384; 6.65%

5211; 90.20%

0

2000

4000

Cou

nt

No Quan Values Shared <Not Set>

Peptide Groups - Quan Info

Quan Results- Details peptides

490; 2.84%

1353; 7.84%

15409; 89.32%

0

5000

10000

15000

Cou

nt

Filtered by Isolation Interference

Filtered by av. S/N

<Not Set>

Quan Spectra - Quan Info Details

All Quan Spectra Peptide groups

Quantified Spectra

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QC Tools- Instrument

Mass Accuracy MS2 Injection Time, Front Optics, LC

Reporter Ions S/N; Quad Status

Example Plots for Lumos SPS, 50 min QC run

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QC Tools Instrument: Housekeeping Proteins Ratios Should be a Perfect 1

• Scaled , • No normalization • Correction factors applied

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QC Tools- Instrument

Mass Accuracy MS2 Injection Time, Front Optics, LC

Fusion SPS data, instrument needs maintenance

Reporter Ions S/N; Quad Status

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QC Tools Instrument: Housekeeping Proteins Ratios Should be a Perfect 1

• Scaled , • No normalization • Correction factors applied

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Yeast TKO TMT 11 Standard: QC run Expected Average Instrument Performance

5587 6604

7490

9092 8734 9045

0

2000

4000

6000

8000

10000

12000

FusionSPS

LumosSPS

FusionMS2

LumosMS2

QE HF QE HF X

Unique Peptides, 50 min run

Identified

Quantified

1147 1320

1495 1682 1623 1649

0200400600800

100012001400160018002000

FusionSPS

LumosSPS

FusionMS2

LumosMS2

QE HF QE HF X

Protein Groups, 50 min run

Identified

Quantified

50

60

70

80

90

100

LumosSPS,MF

LumosSPS

LumosMS2

QE HF QE HFX, TMTmode

Inte

rfere

nce

Free

Inde

x

Met6/parental

His4/parental

Ura2/parental

MF= SPS Mass Matches (%), 65% New feature in PD 2.3

Accuracy of Quantitation, 50 min runs

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Yeast TKO TMT 11 Standard: QC run Expected Average Instrument Performance

0.7

5587 6604

7490

9092 8734 9045

0

2000

4000

6000

8000

10000

12000

FusionSPS

LumosSPS

FusionMS2

LumosMS2

QE HF QE HF X

Unique Peptides, 50 min run

Identified

Quantified

1147 1320

1495 1682 1623 1649

0200400600800

100012001400160018002000

FusionSPS

LumosSPS

FusionMS2

LumosMS2

QE HF QE HF X

Protein Groups, 50 min run

Identified

Quantified

Accuracy of Quantitation, Lumos SPS 50 min run

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Yeast TKO TMT 11 Standard 2 hour run – Method Development/Optimization RT: 0.00 - 185.00

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

44.27416.7878

40.09502.8124

53.52416.788136.49

438.2910110.23

522.994171.29

426.293453.58416.788424.26

482.2881 80.63728.9026

63.69631.8745 139.32

965.0892150.46

679.716180.72

728.9031106.88

671.4331

112.15629.8612 126.74

676.399595.33

695.8983139.39

643.729198.57

611.3865 153.341012.2199131.94

611.3294 140.95889.4877 153.43

836.5388

24.04604.4940

7.60750.4326

153.48929.9942 169.27

445.1208

NL:7.09E9Base Peak MS TKOTT11_1ms2_2hr1

SPS

P15992, Heat Shock protein 26(301ppm)

MS2

1.8Th MS2

Met6/parental>2

Met6/parental<2

Met6/parental<2

2000

3024 2931

0

500

1000

1500

2000

2500

3000

3500

Lumos SPS Lumos MS2 Lumos MS2 (1.8)

Protein Groups, 120 min gradient

Identified

Quantified

10992

17516 16731

0

4000

8000

12000

16000

20000

24000

Lumos SPS Lumos MS2 Lumos MS2(1.8)

Unique Peptides, 120 min gradient

Identified

Quantified

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Summary: Product Information- Available October 1

• Excellent tool for LC and mass spec method development

• Excellent QC assay tool for quality assessment of LC and mass spec instrument status

• Provides accuracy, precision and dynamic range assessments for different mass spec strategies

• Optional: TMT labeled Peptide Retention Time Calibration mix (PRTC, yeast heavy isotope peptides) can be spiked in for triggered, multiplexed assay (TOMAHAQ) method development

• A40938 Pierce TMT11plex yeast digest

standard, 20μg • A40939 Pierce TMT11plex yeast digest

standard, 5 x 20μg

met6Δ his4Δ ura2Δ BY4742

Pierce TMT11 yeast TKO standard

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TOMAHAQ Method Development

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• Jae Choi • Aaron M. Robitaille • John C. Rogers • Kay Opperman • David Horn • Devin Drew • Tabi N. Arrey • Andreas Huhmer

Acknowledgments

Quantitative Labeling Using Tandem Mass...

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