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Rosa Viner Omics marketing group September 13, 2018
Quantitative Labeling Using Tandem Mass Tags
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Introduction: TMT based relative quantification
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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™
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How Does Isobaric Mass Tagging ( TMT&iTRAQ) Work?
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TMT Labels are Indistinguishable
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The Difference Only Appears in MSn
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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
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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 *
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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
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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
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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
11
TMT11 – 131C
Select for MS2 Identification Quantitation
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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]
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
180
200
Ab
un
da
nce
[a
.u.]
96104
159155155
131516
146147147
P00815: histidine biosynthesis trifunctional protein [OS=Saccharomyces cerevisiae S288C]
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.]
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
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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
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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
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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
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
Solvent A: 0.1% formic acid
Solvent B: 80 % Acetonitrile, 0,1% formic acid
Flow rate: 300 nL/min
Injection volume: 1-2 µL
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
Solvent A: 0.1% formic acid
Solvent B: 100 % Acetonitrile, 0,1% formic acid
Flow rate: 300 nL/min
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
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 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
Resolution Full MS 120000 120000 120000 120000
AGC target Full MS 4e5 4e5 4e5 4e5
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
MS2 AGC target 1e4 1e4 1e5 1e5
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
Resolution Full MS 120000 120000 120000 120000
AGC target Full MS 4e5 4e5 4e5 4e5
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
MS2 AGC target 1e4 1e4 1e5 1e5
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
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PD 2.2 workflow set up
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I. Quantification Method Add Lot Specific Correction Factors
1
2
3
4
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
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The world leader in serving science
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
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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