TMT Multiplexing Workflow

TMT multiplexing workflows set the standard for accurate and precise proteomic quantitation, combining advanced multiplexed labeling with high-resolution mass spectrometry. Using isobaric chemical tags, peptides from different samples are chemically identical during chromatographic separation and MS1 detection, eliminating variability caused by retention time shifts or ionization efficiency.

During fragmentation, reporter ions are released and measured with exceptional mass accuracy using state-of-the-art Orbitrap mass spectrometers. This ensures reliable differentiation between closely spaced reporter masses. Advanced acquisition strategies, such as SPS MS³ with Real-Time Search, further minimize ratio compression and interference, guaranteeing that measured intensities accurately reflect true biological differences. With the ability to quantify up to 35 samples in a single experiment, TMT workflows deliver robust, reproducible results. This level of precision provides the confidence and statistical power required for large-scale comparative proteomics, empowering researchers to uncover meaningful biological insights.

Quantitative accuracy of TMT multiplexing workflow. Quantification of protein standards in HeLa proteome background. Six protein digest standard (equimolar) was spiked into a background of HeLa cell lysate digest at 100, 200, 400, and 800 fmol per μg of HeLa digest. Samples were labeled with TMTpro 32plex reagents, pooled in equal amounts, and analyzed using ral-time search with SPS MS³ acquisition method. Quantification of β-galactosidase is shown.

Quantitative precision of TMT multiplexing workflow. The violin plot on the right shows the distribution of coefficients of variation (CVs) for TMT protein quantification using real-time search–based SPS MS³. The violin plot corresponds to ratio group of the two-proteome model shown on the left. Narrower violin widths and tighter distributions indicates quantitative precision across replicates.

TMT multiplexing workflow is a powerful tool for simultaneous identification and quantitation of proteins in multiple sample sets. The multiplexed quantification workflows enable the analysis of up to 35 samples in a single LC-MS experiment.

Multiplexing 35 samples in a single analysis. In a TMT multiplexing quantitation experiments, different isobaric tags label various systemic conditions, and all samples are mixed and analyzed in a single LC-MS experiment. The tags have identical chemical properties, causing all peptides from different TMT-labeled samples to co-elute during LC separation and be detected as a single precursor ion peak in the mass spectrometer. Fragmentation generates unique reporter ions for each condition, allowing peptide identification and quantitation by comparing their intensities.

Proteomics analysis by mass spectrometry often requires small sample amounts due to the limited availability of biological materials like tissues or cell cultures. The TMT multiplexing workflow is highly sensitive, allowing for the detection and analysis of proteins at very low concentrations. Advances in Thermo Scientific Orbitrap mass spectrometers have significantly improved the efficiency and accuracy of proteomic analyses, enabling meaningful results from minimal sample quantities, down to low picogram levels or even from single cells.

Single-cell proteomics using TMT multiplexing. Protein groups identified from 16 different single Hela cell in a single LC-MS analysis.