brochure: tmtpro label reagents — higher multiplex quantitation … · 2019-09-06 · missing...
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TMTpro Label Reagents—higher multiplex quantitation for up to 16 samplesNext generation of TMT labeling reagents for higher throughput and greater quantitative accuracy for multiplexed proteome analysis
TMTpro 16plex Label Reagents
Next-generation TMT reagent set enabling 16plex sample multiplexingThermo Scientific™ TMTpro™ 16plex label reagents are the next generation of tandem mass tags designed to increase the level of sample multiplexing without compromising on protein identification and quantitation.
While Thermo Scientific TMTpro label reagents have a different chemical structure and are ~20% larger in mass than Thermo Scientific TMT reagents, the sample processing and analysis workflow (Figure 2) remains the same, with the added advantage of increased multiplexed quantitation, increased sample throughput, and fewer missing quantitative values among samples.
A
B
Figure 1. Chemical structure of the Thermo Scientific TMTpro label reagents. (A) Functional regions of the reagent structure, including MS/MS fragmentation site by higher-energy collision dissociation (HCD). (B) TMTpro 16plex reagent structures and 13C and 15N stable isotope positions (*).
Massreporter
Massnormalizer
Cleavablelinker
TMTpro-126
TMTpro-127C
TMTpro-128C
TMTpro-129C
TMTpro-130C
TMTpro-131C
TMTpro-132C
TMTpro-133C
TMTpro-127N
TMTpro-128N
TMTpro-129N
TMTpro-130N
TMTpro-131N
TMTpro-132N
TMTpro-133N
TMTpro-134N
Amine-reactivegroup
Highlights• Multiplex—concurrent MS analysis of up to 16 samples
derived from cells, tissues, or biological fluids
• Robust—increased multiplex capability results in fewer missing quantitative values among samples and within replicates for better quantitative accuracy and precision
• Efficient—amine-reactive, NHS ester–activated reagents ensure efficient labeling of all peptides regardless of protein sequence or proteolytic enzyme specificity
• Compatible—optimized for use with high-resolution Thermo Scientific™ Orbitrap™ platforms, including the new Orbitrap Eclipse™ Tribrid™ and Orbitrap Exploris™ 480 mass spectrometers with data analysis fully supported by Thermo Scientific™ Proteome Discoverer™ 2.4 software
The original Thermo Scientific™ Tandem Mass Tags (TMT) were designed to enable multiplexing of up to 11 samples in a single LC-MS analysis without any changes in reagent structure or molecular weight.
The new TMTpro tag is similar in design to TMT as it is isobaric and amine reactive, but differs in structure with a longer spacer region and isobutyl proline mass reporter region (Figure 1). After MS/MS fragmentation, each TMTpro tag generates a unique reporter mass (126–134 Da) in the low-mass region of the high-resolution MS/MS spectrum that is used for relative quantitation of protein expression levels.
2,914
5,473
2,874
5,644
0
1,000
2,000
3,000
4,000
5,000
6,000
Beforefractionation
Afterfractionation
Beforefractionation
Afterfractionation
Beforefractionation
Beforefractionation
Afterfractionation
TMT11plex TMTpro 16plex
Pro
tein
iden
tifica
tion
16,91114,525
43,078
05,000
10,00015,00020,00025,00030,00035,00040,00045,00050,000
TMT11plex TMTpro 16plex
Pep
tide
iden
tifica
tion
Afterfractionation
44,962
New TMTpro 16plex label reagents have the same labeling effi ciency and peptide/protein ID rates as TMT reagents (Figures 3 and 4). The TMTpro reagents provide the same level of quantitative precision as the original TMT reagents,
Figure 4. Unique protein and peptide identifi cations of HeLa cell digest labeled with TMT11plex or TMTpro reagents analyzed by LC-MS before or after high pH reversed-phase fractionation.
Figure 5. Venn diagrams of overlapping proteins quantifi ed among 30 replicate HeLa cell digests labeled with three TMT11plex reagent sets or two TMTpro 16plex reagent sets.
TMTpro 126
TMTpro 127N
:
:
:
TMTpro 134N
Label
Sample 1
Sample 2
:
:
:
Sample 16
Treatsamples
and isolateproteins
Denature,reduce,
alkylate, andtryptic digest
Fractionate,clean up,perform
LC-MS/MSanalysis
Data analysis and quantitation
using Proteome Discoverer Software
Combine
Figure 2. Procedure summary for MS experiments using TMTpro isobaric mass tagging reagents. Protein extracts from cells or tissues are reduced, alkylated, and then digested using the EasyPep™ Mini MS Sample Prep Kit or an equivalent method. Samples are then labeled with the TMTpro reagents before combining, fractionating, and cleaning up. Labeled samples are analyzed on a high-resolution Orbitrap LC-MS/MS mass spectrometer before data analysis to identify peptides and quantify relative abundance of reporter ions.
with the benefi t of improved quantitative accuracy with larger sample sets. Higher-multiplex tags also have fewer missing values among replicates, resulting in up to 20% more quantifi able proteins in 50% less time (Figure 5).
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%TMTpro 16plex TMT11plex
TMTp
ro-12
6
TMTp
ro-12
7N
TMTp
ro-12
7C
TMTp
ro-12
8N
TMTp
ro-12
8C
TMTp
ro-12
9N
TMTp
ro-12
9C
TMTp
ro-13
0N
TMTp
ro-13
0C
TMTp
ro-13
1N
TMTp
ro-13
1C
TMTp
ro-13
2N
TMTp
ro-13
2C
TMTp
ro-13
3N
TMTp
ro-13
3C
TMTp
ro-13
4N
TMT-1
26
TMT-1
27N
TMT-1
27C
TMT-1
28N
TMT-1
28C
TMT-1
29N
TMT-1
29C
TMT-1
30N
TMT-1
30C
TMT-1
31N
TMT-1
31C
Figure 3. HeLa cell digests were individually labeled with Thermo Scientifi c™ TMTpro or TMT11plex™ reagents and analyzed by LC-MS to determine labeling effi ciency of peptide amino groups (lysine and N termini).
Found in fi les: [F44]147
Found in fi les: [F45]174
197
169 166
232Found in fi les: [F46]
TMT11plex
TMTpro 16plex
2896
3168Found in fi les: [F43]
344Found in fi les: [F42]
366
Find out more at thermofisher.com/tmtproFor Research Use Only. Not for use in diagnostic procedures. © 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TMT11plex and TMTpro are trademarks of Proteome Sciences. Tandem Mass Tag is a trademark of Electrophoretics Limited. COL23594 0919
Applications• Protein identification and quantitation from multiple
samples of cells, tissues, or biological fluids
• Protein expression profiling of normal vs. abnormal states, or control vs. treated cells
• Quantitative analysis of proteins for which no antibodies are available
Ordering information
Product Quantity Cat. No.
Sample preparation
EasyPep Mini MS Sample Prep Kit 20 reactions A40006
EasyPep Maxi MS Sample Prep Kit 8 reactions A45734
EasyPep 96 MS Sample Prep Kit 96 reactions A45733
Pierce Trypsin/Lys-C Mix, MS grade 20 µg A40007
Pierce Trypsin/Lys-C Mix, MS grade 5 x 20 µg A41007
Pierce Trypsin/Lys-C Mix, MS grade 100 µg A40009
Pierce High pH Reversed-Phase Fractionation Kit 1 kit 84868
High-Select Fe-NTA Phosphoenrichment Kit 1 kit A32992
High-Select TiO2 Phosphoenrichment Kit 1 kit A32993
High-Select HSA/Immunoglobulin Depletion Mini Spin Columns 6 columns A36366
High-Select Top14 Abundant Protein Depletion Mini Spin Columns 6 columns A36370
Protein quantitation
TMTpro 16plex Isobaric Label Reagent Set (sufficient for one 16plex isobaric experiment)
1 x 0.5 mg A44521
TMTpro 16plex Isobaric Label Reagent Set (sufficient for six 16plex isobaric experiments)
6 x 0.5 mg A44522
TMTpro 16plex Isobaric Label Reagent Set (sufficient for ten 16plex isobaric experiments)
1 x 5 mg A44520
TMTpro-Zero Isobaric Label Reagent (sufficient for labeling five samples) 5 x 0.5 mg A44519
TMTpro-Zero Isobaric Label Reagent (sufficient for labeling ten samples) 1 x 5 mg A44518
Standards
Pierce TMT11plex Yeast Digest Standard 20 µg A40938
Pierce TMT11plex Yeast Digest Standard 5 x 20 µg A40939
• Identification and quantitation of membrane and posttranslationally modified proteins
• Identification and quantification of hundreds to thousands of proteins in a single experiment