TMTpro Label Reagents—higher multiplex

quantitation for up to 16 samples

Next 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 multiplexing A
Thermo 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. Mass Mass Amine-reactive
reporter normalizer group
Cleavable
linker
B

TMTpro-126 TMTpro-127N

TMTpro-127C TMTpro-128N

Highlights
• Multiplex—concurrent MS analysis of up to 16 samples TMTpro-128C TMTpro-129N

derived from cells, tissues, or biological fluids

• Robust—increased multiplex capability results in fewer TMTpro-129C TMTpro-130N

missing quantitative values among samples and within
replicates for better quantitative accuracy and precision
TMTpro-130C TMTpro-131N
• Efficient—amine-reactive, NHS ester–activated reagents
ensure efficient labeling of all peptides regardless of
TMTpro-131C TMTpro-132N
protein sequence or proteolytic enzyme specificity

• Compatible—optimized for use with high-resolution

TMTpro-132C
Thermo Scientific™ Orbitrap™ platforms, including the TMTpro-133N

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.
TMTpro-133C TMTpro-134N
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 (*).

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.
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.
 Label Combine Fractionate, Data analysis and
Sample 1 clean up, quantitation
TMTpro 126
Treat Denature, perform using Proteome
Sample 2 samples reduce, LC-MS/MS Discoverer Software
TMTpro 127N
and isolate alkylate, and analysis
: proteins tryptic digest :

: :

: :

Sample 16 TMTpro 134N

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.

New TMTpro 16plex label reagents have the same labeling with the benefit of improved quantitative accuracy with
efficiency and peptide/protein ID rates as TMT reagents larger sample sets. Higher-multiplex tags also have fewer
(Figures 3 and 4). The TMTpro reagents provide the same missing values among replicates, resulting in up to 20%
level of quantitative precision as the original TMT reagents, more quantifiable proteins in 50% less time (Figure 5).

TMTpro 16plex TMT11plex

100.0%
80.0%
60.0%
40.0%
20.0%
0.0%
TM 126
6

TM 0N

TM 0C
TM 9N

TM 9C
TM 8N

TM 8C
T M 7N

TM 7C

TM 1N

1C
0N

0C

3N

3C
9N

9C

T M 4N
8N

2N

2C
8C
7N

7C

1N

1C
12

13

13

13

13
12

12

12

12

12

12
T-
TM -13

13

TM -13

TM -13

TM -13

TM -13

TM -13

TM -13

3
12

12

TM -12

TM -12

12

TM -12
o-

-1

T-

T-

T-

T-

T-

T-

T-

T-

T-

T-
o-

o-

-
r

ro

ro

ro

ro

ro

ro

ro

ro

ro

ro

ro

ro

ro
Tp

r
Tp

Tp

Tp

Tp

Tp

Tp

Tp

Tp

Tp

Tp

Tp

Tp

Tp

Tp

Tp
TM

TM

TM

TM

TM

TM

Figure 3. HeLa cell digests were individually labeled with Thermo Scientific™ TMTpro or TMT11plex™ reagents and analyzed by LC-MS to determine
labeling efficiency of peptide amino groups (lysine and N termini).

6,000 5,644
5,473 197
Found in fi les: [F44] Found in fi les: [F45]
5,000
Protein identification

147 174
4,000
2,914 2,874
3,000

2,000 2896
1,000 169 166
0
Before After Before After
fractionation fractionation fractionation fractionation 232
TMT11plex
TMT11plex TMTpro 16plex Found in fi les: [F46]
50,000
44,962 43,078
45,000
Peptide identification

40,000
35,000
30,000
25,000
20,000 16,911
15,000
14,525 3168
Found in fi les: [F42] Found in files: [F43]
10,000 344
366
5,000
0
Before After Before After
fractionation fractionation fractionation fractionation TMTpro 16plex
TMT11plex TMTpro 16plex

Figure 4. Unique protein and peptide identifications of HeLa cell Figure 5. Venn diagrams of overlapping proteins quantified among
digest labeled with TMT11plex or TMTpro reagents analyzed by 30 replicate HeLa cell digests labeled with three TMT11plex reagent
LC-MS before or after high pH reversed-phase fractionation. sets or two TMTpro 16plex reagent sets.
Applications
• Protein identification and quantitation from multiple • Identification and quantitation of membrane and
samples of cells, tissues, or biological fluids posttranslationally modified proteins

• Protein expression profiling of normal vs. abnormal • Identification and quantification of hundreds to thousands
states, or control vs. treated cells of proteins in a single experiment

• 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
1 x 0.5 mg A44521
(sufficient for one 16plex isobaric experiment)
TMTpro 16plex Isobaric Label Reagent Set
6 x 0.5 mg A44522
(sufficient for six 16plex isobaric experiments)
TMTpro 16plex Isobaric Label Reagent Set
1 x 5 mg A44520
(sufficient for ten 16plex isobaric experiments)
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

Find out more at thermofisher.com/tmtpro

For 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