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TMT10plex™ Total Proteome profiling service provides accurate global profiling of protein abundance in cells and tissues, using multiplexed sample labeling with Tandem Mass Tags™ (TMT™) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Together, these technologies enable proteomics-driven discovery, biomarker analysis, and mechanism-of-action studies to be carried out with greater depth of coverage while delivering high quality, quantitative data on thousands of proteins with predictable and reliable turnaround times.

Instrumentation

TMT10plex Total Proteome services are performed on a Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer, which provides the highest number of protein identifications and the most accurate quantitation compared to other instrumentation. CST is one of the first licensed commercial providers of TMT10plex™ proteomic services in the U.S.

Sample Prep

Sample preparation is easy. Just send frozen cell or tissue samples (just 100 μg total protein per sample) and our team will do the rest. Total Proteome services are available by assay runs of 10 samples; for projects involving >10 samples, one of the channels is reserved for a control peptide mix.

Multiplexing with TMT™

TMT™ labels are isobaric mass tags that use amine-reactive chemistry to label all peptides present in a digested sample. Thousands of proteins may be routinely detected and quantified in each study. Samples (100 μg input protein each) are uniquely labeled, combined into a single sample, fractionated, and run in LC-MS/MS, offering significant flexibility for experimental design. Studies greater than 10 samples can be analyzed using a control reference mixture in each TMT10plex™ assay, allowing projects of any size to be run.

Experimental design approaches may include:

  • Tumor profiling
  • Compound library screening
  • Dose dependence studies
  • Time-course studies
  • Cell line, primary cell type, or tissue type comparison
  • Ligand, agonist, and/or antagonist analyses

What You Get

CST’s proteomics scientists partner with our clients to provide defined timelines and consultative support throughout the project, from pre-study planning to data analysis and follow-up. Clients receive a comprehensive data package including:

  • Results table and slide deck containing protein identifications and TMT™ quantitation.
  • Post-study consultation.
  • Optional mapping of TMT10plex™ Total Proteome results to canonical pathways and new interaction networks using Qiagen’s Ingenuity® Pathway Analysis (IPA) software (available as an additional service).

Contact us for further information on TMT10plex™ Proteomic Services.

Note: TMT10plex™ Total Proteome service is available for U.S.-based customers only.

Cell Signaling Technology and CST are trademarks of Cell Signaling Technology, Inc.

Thermo, Thermo Fisher, Thermo Fisher Scientific, Fusion, Lumos, and Tribrid are licensed trademarks of Thermo Fisher Scientific and its subsidiaries.

Tandem Mass Tag, TMT, and TMT10Plex are licensed trademarks of Electrophoretics Limited.

Ingenuity is a licensed trademark of Qiagen.

These proteomic services and resulting deliverables are sold under an agreement for Tandem Mass Tag™ (TMT™) technology between Pierce Biotechnology, Inc and Cell Signaling Technology, Inc. and are provided for internal research puposes only. No rights for clinical activities or studies or for any Commercial Purpose are conveyed hereunder. In no event will the producer or distributor of TMT™ technology be liable for incidental, special, multiple or consequential damages in connection with or arising from the use of these services or deliverables.

“Commercial Purposes” means any (i) use for the analysis of nucleic acids for genomic purposes; (ii) use, directly or indirectly, in manufacturing, production, or quality control; (iii) use to provide a service, information, or data for consideration; (iv) use for therapeutic, diagnostic or prophylactic purposes; or (v) sale, resale, leasing, or licensing, whether or not for research purposes. For purposes of clarification, Commercial Purpose does not include use to provide information or data to an end-user’s subcontractors or its contract research organizations that are conducting research on behalf of said end-user.

For information on acquiring rights for any use not permitted herein, please contact Pierce Biotechnology outlicensing at outlicensing@lifetech.com.

TMT™ Labeling and Detection

TMT Thermo

Each digested protein sample is labeled with a unique TMT™ tag. Samples are fractionated, purified, and then subject to liquid chromotography. (Image courtesy of Thermo Fisher Scientific).

BRP Fractionation

Fractions from basic reverse-phase high performance liquid chromatography (bRP-HPLC) are collected and non-sequentially pooled prior to performing MS, contributing to improved depth of coverage.

Multi-notch MS3 quantitation, available only on Orbitrap™ Fusion™ systems, offers unparalleled accuracy of TMT™ quantitation. Multi-notch MS3 avoids the pitfall of underestimated fold changes in protein expression caused by contaminant reporter ion interference that occurs in traditional MS2 methods (1, 2). This is accomplished by synchronous precursor selection (SPS) and coisolation/cofragmentation of multiple MS2 fragments, increasing precursor ion capture, signal intensity, dynamic range, and accuracy of analysis. Ultimately, this means more proteins can be identified and accurately quantified.

TMT10plex™ Proteome Analysis in Cell Lines

14 human cell lines commonly used in labs around the world were analyzed using TMT10plex™. A mixture of peptides from all 14 cell lines (numbered below) was used to standardize the quantitation across multiple 10-plexed experiments. Relative protein abundance in each cell line compared to the control mix was calculated and color-coded to reflect proteins that were higher abundance than control (green) or lower abundance compared to control (red).

Slide 1 Heatmap

Heat map of color-coded relative protein abundances measured with TMT10plex™ across 14 human cell lines (numbered). Over 8000 proteins were quantified (upper), and a subset of data points shown with color coding and % change in abundance relative to control mix (lower).

Clients can view data globally using hierarchical clustering or other tools, and can review individual protein data in the tables.

TMT10plex™ data was compared to to western blots (WB) to validate protein abundance measurements. TMT™ quantitation shows good agreement with WB. FoxA2 and CD3ε expression levels varied with cell line, while Erk1 and Erk2 exhibited less cell line-dependent variation in expression.

TMT Western Blot

Relative protein abundance quantitation in 14 human cell lines (numbered) by TMT10plex™ profiling (bar graphs) and aligned western blots for FoxA2 (upper), CD3ε (middle) and Erk1/2 (lower).

These data demonstrate the ability of the TMT10plex™ Total Proteome method to provide detailed, quantitative information on relative protein abundance from multiplexed samples.

  1. Ting L, Rad R, Gygi SP, Haas W (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat. Methods 8(11), 937–40.
  2. McAlister GC, Nusinow DP, Jedrychowski MP, Wühr M, Huttlin EL, Erickson BK, Rad R, Haas W, Gygi SP (2014) MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem. 86(14), 7150–8.