Thermo Scientific™ TMTpro 18-plex, TMTpro-134C, and TMTpro-135N Label Reagents

Product Code.	Quantity	Label or Dye	unitSize
17496572	1 x 5 mg	TMTpro 18-plex	5mg
17331783	1 x 5 mg	TMTpro-134C and TMTpro-135N	5mg
17418273	6 x 0.5 mg	TMTpro 18-plex	3mg
17423772	6 x 0.5 mg	TMTpro-134C and TMTpro-135N	3mg

Product Code. 17496572 Supplier Thermo Scientific™ Supplier No. A52045

Description

TMTpro 18plex Label Reagents enable multiplexing of up to 18 samples. Addition of TMTpro 134C and TMTpro 135N Label Reagents increase sample multiplexing from 16-plex to 18-plex, enabling even greater throughput for protein identification and quantitation using tandem mass spectrometry (MS).

TMTpro 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. The Thermo Scientific TMTpro 18plex Label Reagent Set enables multiplexing up to 18 samples, enabling even greater throughput for protein identification and quantitative analysis by tandem mass spectrometry (MS).

The structures of the TMTpro and TMT tags are similar in being isobaric and amine reactive, but the TMTpro tag has a longer spacer region and isobutyl proline mass reporter. After MS/MS fragmentation, each TMTpro 18plex tag generates a unique reporter mass (e.g., TMTpro 126-135 Da) in the low-mass region of the high-resolution MS/MS spectrum that is used for relative quantitation of protein expression levels.

The TMT pro134C and TMTpro135N Label Reagents are identical in composition and structure to the other TMTpro 16plex tags, but are not isobaric. Addition of the TMTpro 134C and 135N tags create a 17th and 18th channel for relative quantitation using high resolution Orbitrap MS instruments. These tags are available separately (Cat. No. A52046, A52048), as part of the TMTpro 18plex Label Reagent Set, or as part of the complete 18plex set of reagents in a 96-well plate format.

Features of the TMTpro 18plex Label Reagent Set include:

• Multiplex—concurrent MS analysis of up to 18 samples derived from cells, tissues, or biological fluids
• Robust—increased multiplex capability results in fewer missing quantitative values among samples and higher confidence among replicates
• Efficient—amine-reactive, NHS ester-activated reagents ensure greater than 95% labeling of all peptides regardless of protein sequence or proteolytic enzyme specificity
• Optimized for use—with high resolution Thermo Scientific MS/MS Orbitrap platforms, analysis fully supported by Proteome Discoverer 2.3
• Convenient size—provided in a ready-to-use, single-use format to ensure optimal stability and performance

For high resolution analysis of TMTpro-labeled peptides, the recommended LC column for the Nanospray Flex source is the Acclaim PepMap 100 C18 LC Column (Cat. No. 164942 or 164939). For the EASY-Spray source, the recommended LC column is the EASY-Spray C18 LC Column (Cat. No. ES902 or ES903). When combined with the industry-leading, high resolution Orbitrap instruments and software, TMTpro reagents provide an integrated total solution for quantitative protein expression analysis.

Specifications

• Product Type: Isobaric Label Reagent Set
• Content And Storage: Store at –20°C.
• Label or Dye: TMTpro 18-plex
• Starting Material: Peptides
• Detection Method: Mass Spectrometry
• Workflow Step: Peptide labeling and quantitation
• Quantity: 1 x 5 mg

Frequently Asked Questions (FAQs)

Can I combine TMTpro tags with TMT tags?

We do not recommend mixing TMTpro and TMT tags, as they differ in chemical structure.

Can I purchase the TMTpro-134C and TMTpro-135N Label Reagents separately from the TMTpro 18-plex Label Reagent Set?

Yes, Cat. No. A52046 provides TMTpro-134C and TMTpro-135N tags as 1 vial each of 5 mg and Cat. No. A52048 provides TMTpro-134C and TMTpro-135N tags as 6 vials each of 0.5 mg.

What are the components of the TMTpro 18-plex Label Reagent Sets?

The TMTpro 18-plex Label Reagent Sets are the combination of corresponding TMTpro 16 plex Label Reagent Sets and TMTpro-134C & TMTpro-135N Label Reagents. Cat. No. A52045 is a combination of Cat. No. A52046 and Cat. No. A44520 where all TMTpro tags are provided as 1 vial each of 5 mg. Cat. No. A52047 is a combination of Cat. No. A52048 and Cat. No. A44522 where all TMTpro tags are provided as 6 vials each of 0.5 mg.

At which step should I label my mass spec samples with TMT reagents?

TMT labeling can be performed after reduction, alkylation, digestion, or prior to cleanup, if the sample is buffered at appropriate pH (8-8.5) in a suitable buffer without primary amines (e.g., Tris, glycine). TMT labeling can also be performed after peptide clean-up. Labeling of peptides after clean-up enables measuring and normalizing of the peptide samples for equal mixing.

Do I have to apply correction factors for analyzing data for TMT/TMTpro-labeled samples?

Applying correction factors is required for more accurate relative protein quantitation as the isotopic impurity is variable among the tags, ranging from 5-10%.

How do I bridge the data from different sample sets for TMT-based quantitation?

This is most often done with the utilization of a "pool channel" for each multiplex set. This reference channel typically contains an equimolar mixture of all samples which is labeled with one of the TMT tags that is shared among the sets. The pool channel can be used to then normalize relative protein abundance measurements across multiplexed sets.

In the EasyPep sample preparation workflow, it is mentioned that TMT/TMTpro reagent can be added to peptides before or after the clean-up step. Are there any differences between these two methods, regarding the labeling efficiency or relative quantitation?

The EasyPep kit chemistry is fully compatible with TMT/TMTpro reagents. Most commonly, TMT/TMTpro reagents are used to label peptides immediately after digestion and before peptide cleanup. This enables combining samples for a single cleanup step which reduces variability for more reproducible quantitative measurements. Another option is to label samples after peptide cleanup. This approach provides efficient labeling of the samples but may require the use of a peptide quantification assay to ensure that samples are equally mixed before LC-MS analysis. Both the workflows should provide high labelling efficiencies using our recommended TMT/TMTpro reagent to sample ratios.

What type of mass spectrometer can I use for Tandem Mass Tag (TMT) analysis?

We recommend using high-resolution Orbitrap Tribrid (e.g., Fusion, Lumos, Eclipse), Orbitrap Exploris (e.g., 240, 480), or Q Exactive (e.g,. Plus, HF, HFX) series of instruments.

What is the main purpose of Tandem Mass Tag (TMT) reagents?

Tandem Mass Tag technology is used to individually label different protein samples so they can be combined into a single sample for LC-MS analysis. The major advantages of this workflow are higher sample throughput, less instrument analysis time, high precision of peptide quantitation among replicates, and fewer missing quantified proteins among different samples.

Is TMT quantitation relative or absolute?

TMT quantitation is relative between samples unless a heavy peptide standard is included for comparison.

What is the main purpose of TMT (Tandem Mass Tag) reagents?

TMT technology is used to perform multiplex quantitation of proteins extracted from cells and tissues. Rather than quantify single proteins (for example with an antibody), here, large numbers of proteins can be quantified in a single run.

When should I use quadrupole versus ion trap isolation?

Quadrupole isolation is faster because ions must travel through the quadrupole analyzer regardless of the analysis type chosen. As a result, there is little to no time penalty when using the quadrupole for MS² isolation. In such a case, quadrupole transmission efficiency will depend on isolation width, m/z, and the cleanliness of the analyzer. Ion trap isolation is best suited for MS³ experiments, including those performed using multinotch isolation for TMT analysis; these types of isolation events are not possible with the quadrupole analyzer.

How effective is the salt removal using Pierce Peptide Desalting Spin Columns?

With Pierce Peptide Desalting Spin Columns, more than 90% of salt or labeling tags such as TMT tags can be removed.

What is the reproducibility of Pierce Peptide Desalting Spin Columns from sample to sample?

With Pierce Peptide Desalting Spin Columns, the reproducibility coefficient of variation (CV) is ± 20%.