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