Proteomics techniques are evolving to become a highly sensitive, quantitative, and high-throughput approach to analyzing global protein dynamics within a cell, tissue or an organism. In the past decade, critical advances have been made across the entire proteomics workflow, most notably the development of Tandem Mass Tag Reagents (TMT) and state-of-the-art advancements in mass spectrometry, which have enabled more accurate, high-throughput (comparison of up to 18 samples in a single mass spectrometry analysis) quantitative proteomics.
Quantitative proteomics review: Magic of multiplexing in LC-MS
Learn how TMT proteomics technology and the magic of multiplexing are helping scientists follow proteins in a cell over time to better understand what makes a cell functional, why and how molecules move about in a cell and what the practical applications are to this type of work.
How TMT quantitative proteomics works
TMT are isobaric chemical tags that provide multiplexing capabilities for relative quantitative proteomics analysis. This ability to perform concurrent mass spectrometry (MS) analysis of multiple samples increases throughput and enables relative quantitation of up to 18 different samples derived from cells, tissues or biological fluids. The tags consist of an MS/MS reporter group, a spacer arm and an amine reactive group. The amine reactive group binds to the N- terminus of a peptide or to a lysine residue during labeling.
In relative quantitation experiments, different isobaric tags are used to label different systemic conditions. Once labeled, all samples are mixed and analyzed in a single liquid chromatography-mass spectrometry (LC-MS) experiment. Because the isobaric tags possess the same chemical properties, all peptides from different TMT-labeled samples co-elute during LC separation. Once the peptides enter the mass spectrometer, they are detected simultaneously as a single and indistinguishable precursor ion peak.
Click image to enlarge
Following fragmentation, the tags from each individual systemic condition generate a unique signature reporter ion in the low m/z area of the MS/MS spectrum. Peptide identification is achieved by matching the resulting ion peaks to those provided in fragment databases. Peptide quantitation is accomplished by comparing the intensities of the reporter ions.
The Thermo Scientific Orbitrap Astral Mass Spectrometer delivers accurate and reproducible quantitation of thousands of proteins. Though the novel design of the Orbitrap Astral mass spectrometer, the instrument sets a new standard for TMT analysis to dig deeper into the proteome in record time. Since the Astral analyzer combines high speed and sensitivity with high resolution, it can readily resolve TMT reporter ions for multiplexed quantitation.
The parallelization of the Orbitrap and Astral analyzers transforms TMT analysis by dramatically increasing the depth of coverage and throughput.
Ideally suited for highly complex samples
Thermal profiling
Whole proteome profiling
Whole cell interactomics
Subcellular localization
Highest throughput and coverage
High quantitative precision
Entry level MS/MS system
Obtain maximum quantitative insights from untargeted proteome profiles to targeted proteomics experiments with industry leading single-cell sensitivity and extraordinary accuracy, precision and simplicity. With curated workflows that deliver greater usability, the Thermo Scientific Orbitrap Exploris 480 Mass Spectrometer accelerates your path to large-scale studies, delivering proven high data quality and time savings, so you can go beyond faster to actionable outcomes.
Ideally suited for low to medium sample complexity
Interaction studies with pull downs
Immunoprecipitations
Purified samples
High quantitative precision
Best in class Tribrid mass spectrometer The Thermo Scientific Orbitrap Ascend Tribrid Mass Spectrometer boasts the highest resolving power of all our Orbitrap LCMS instruments (up to 480,000 at m/z 200), achieving extreme detection limits and selectivity in full-scan MS mode. This system offers the exclusive Real-time Search for Synchronous Precursor Selection MS (SPS MS) for highly accurate and precise TMT quantitation results. This unique acquisition strategy improves the ratio accuracy for TMT reporter ions and boosts sensitivity, increasing total quantified peptides. Along with the new QR5 Segmented Hyperbolic-Surface Quadrupole, ion transmission is improved on narrow isolation windows, which is crucial for minimizing interferences. The brighter ion source permits 0.4 amu isolation windows and minimizes ratio inaccuracy.
Ideally suited for highly complex samples
Whole proteome profiling
Whole cell interactomics
Subcellular localization
Highest quantitative accuracy and precision with real-time search enabled MS3 acquisition
Realize high-throughput quantitative proteomics with eleven different isobaric tadem mass tag labels. This set can be used to prepare up to eleven different peptides for parallel labeling and concurrent analysis.
The next generation of tandem mass tags, designed to increase the level of sample multiplexing to enable MS analysis of up to 18 samples without compromising on protein identification and quantitation.
A lyophilized yeast peptide mixture of four congenic strains labeled with TMT11plex reagents to monitor LC-MS/MS system performance.
Perform protein quantitation and identification easily
Isobaric labeling experiments require both qualitative and quantitative data extraction from raw LC-MS files. Thermo Scientific Proteome Discoverer software is the most comprehensive data analysis platform for qualitative and quantitative proteomics research; it not only supports differential analysis of fragmentation data at the SPS MS3 level for protein quantitation, but also at the MS2 level for protein identification. The software offers a comprehensive data mining tool set and supports multiple search engines during peptide identification.
The Thermo Scientific Orbitrap Astral Mass Spectrometer delivers accurate and reproducible quantitation of thousands of proteins. Though the novel design of the Orbitrap Astral mass spectrometer, the instrument sets a new standard for TMT analysis to dig deeper into the proteome in record time. Since the Astral analyzer combines high speed and sensitivity with high resolution, it can readily resolve TMT reporter ions for multiplexed quantitation.
The parallelization of the Orbitrap and Astral analyzers transforms TMT analysis by dramatically increasing the depth of coverage and throughput.
Ideally suited for highly complex samples
Thermal profiling
Whole proteome profiling
Whole cell interactomics
Subcellular localization
Highest throughput and coverage
High quantitative precision
Entry level MS/MS system
Obtain maximum quantitative insights from untargeted proteome profiles to targeted proteomics experiments with industry leading single-cell sensitivity and extraordinary accuracy, precision and simplicity. With curated workflows that deliver greater usability, the Thermo Scientific Orbitrap Exploris 480 Mass Spectrometer accelerates your path to large-scale studies, delivering proven high data quality and time savings, so you can go beyond faster to actionable outcomes.
Ideally suited for low to medium sample complexity
Interaction studies with pull downs
Immunoprecipitations
Purified samples
High quantitative precision
Best in class Tribrid mass spectrometer The Thermo Scientific Orbitrap Ascend Tribrid Mass Spectrometer boasts the highest resolving power of all our Orbitrap LCMS instruments (up to 480,000 at m/z 200), achieving extreme detection limits and selectivity in full-scan MS mode. This system offers the exclusive Real-time Search for Synchronous Precursor Selection MS (SPS MS) for highly accurate and precise TMT quantitation results. This unique acquisition strategy improves the ratio accuracy for TMT reporter ions and boosts sensitivity, increasing total quantified peptides. Along with the new QR5 Segmented Hyperbolic-Surface Quadrupole, ion transmission is improved on narrow isolation windows, which is crucial for minimizing interferences. The brighter ion source permits 0.4 amu isolation windows and minimizes ratio inaccuracy.
Ideally suited for highly complex samples
Whole proteome profiling
Whole cell interactomics
Subcellular localization
Highest quantitative accuracy and precision with real-time search enabled MS3 acquisition
Realize high-throughput quantitative proteomics with eleven different isobaric tadem mass tag labels. This set can be used to prepare up to eleven different peptides for parallel labeling and concurrent analysis.
The next generation of tandem mass tags, designed to increase the level of sample multiplexing to enable MS analysis of up to 18 samples without compromising on protein identification and quantitation.
Perform protein quantitation and identification easily
Isobaric labeling experiments require both qualitative and quantitative data extraction from raw LC-MS files. Thermo Scientific Proteome Discoverer software is the most comprehensive data analysis platform for qualitative and quantitative proteomics research; it not only supports differential analysis of fragmentation data at the SPS MS3 level for protein quantitation, but also at the MS2 level for protein identification. The software offers a comprehensive data mining tool set and supports multiple search engines during peptide identification.
The Thermo Scientific FAIMS Pro Duo interface enhances precursor selectivity, improving qualitative and quantitative results for most peptide and protein applications while reducing time-consuming sample preparation steps. The FAIMS Pro Duo interface increases analytical performance through gas-phase fractionation and selective enhancement of peptidic compounds, reducing the complexity of the MS spectra, and improving analyte signal/noise ratio. The end result is greater proteome coverage, enhanced sensitivity and more accurate quantiation.
Tandem mass tag mass spectrometry advancements for highest quantitative results
Our Orbitrap LC-MS-based TMT quantitation solutions are the most published in the industry compared to other isobaric tagging applications (see Resources section below for articles and documents). To obtain the most complete quantitative profiles we have delivered improvements that enable the highest quantitative accuracy and precision to meet research goals.
Recent improvements have been made both in terms of technology and performance of mass spectrometry as well as taking advantage of intelligent acquisition and algorithms that more intelligently pull out real, quality data from raw spectra. Together with TMT reagents, they deliver the industry’s gold standard for the identification and quantitation of thousands of proteins from multiplexed samples in a single run that is the most accurate, precise and cost effective for functional biology studies.
TurboTMT intelligent acquisition based on novel ΦSDM spectral processing increases resolution to baseline resolve TMT reporter ion isotopologues and speeds up spectral acquisition for TMT 11plex experiments, thereby increasing throughput for identification of quantifiable peptides.
The Precursor Fit filter allows for selecting ions based on precursor ion specificity of the isolation window. This increases precursor ion isolation specificity and decreases the chance of triggering on precursors with a wrong annotation of charge state/monoisotopic peak, or when an isolation window is not specific to the precursor of interest.
Real-Time Search data acquisition improves experimental speed and quantitation accuracy
Real-Time Search improves experimental speed and quantification accuracy when combined with the advanced SPS MS3 experimental approach. With Real-Time Search data acquisition, MS3 scans are only triggered if a peptide-spectrum match (PSM) is identified from the preceding MS2. This reduces the number of MS3 events, increases the precursor sampling rate and results in a greater number of quantified peptides; improving experimental speed. With the aid of the Real-Time Search data acquisition strategy, the Orbitrap Ascend Tribrid MS selects only those fragment ions that correspond to the identified PSM for quantification in the MS3 scan, resulting in improved quantitation accuracy.
Adding the FAIMS Pro interface to the workflow increases precursor selectivity, reduces interference, and gas-phase fractionation resulting in greater accuracy in TMT peptide quantification.
The Thermo Scientific Tandem Mass Tag multiplexing workflow with unique, intelligent acquisition management delivers best-in-class quantification for multiplexed proteome analysis. By leveraging intelligent acquisition schemes built into HRAM Orbitrap MS we enable life scientists to quantify proteins with the highest confidence without sacrificing proteome coverage or depth and with easy set up.
In combination with TMT reagents, we deliver three complete Thermo Scientific Orbitrap MS workflows that include sample preparation for the identification and quantitation of thousands of proteins from multiplexed samples in a single run that is the most accurate, precise and cost effective for functional biology studies. Each workflow is optimized for highest quantitative accuracy/precision, coverage depth and minimizing co-ion interference and ratio compression.
Thermo Scientific TMT MS2 multiplexing workflow with unique, intelligent acquisition management delivers best-in-class quantitation for MS2 based multiplexed proteome analysis yielding unrivaled confidence in discovery proteomics experiments with no compromise in coverage or depth.
The Orbitrap Astral mass spectrometer utilizes novel analyzer technology, delivering high sensitivity and speed (200 Hz) without sacrificing the resolving power to achieve new levels of performance for TMT MS2 multiplexing. The Orbitrap Astral mass spectrometer enables faster throughput with deep proteome coverage for TMT MS2 multiplexing experiment than any commercial instrument on the market. The new analyzer on the instrument provides a resolution of 50,000 in the low m/z region to baseline resolve reporter ions for TMT 18plex experiments.
The TMT MS2 workflow using the Orbitrap Exploris 480 mass spectrometer utilizes intelligent acquisition schemes (TurboTMT mode, PrecursorFit filter) to deliver high number of quantifiable peptides for multiplexed proteome analysis. In combination with TMT reagents, and the FAIMS Pro interface, the Orbitrap Exploris 480 MS is one of two dedicated MS/MS system with resolving power that can perform TMT 18plex experiments yielding unrivaled confidence in discovery proteomics experiments with no compromise in coverage or depth.
The FAIMS Pro interface reduces co-ion interference and improves quantitative accuracy.
Thermo Scientific SPS MS3 multiplexing workflow on Thermo Scientific Orbitrap Tribrid mass spectrometers provides a unique, unmatched capability to accurately measure the subtle changes in proteome for multiplexed proteome analysis.
The use of SPS MS3 compared to MS2 dramatically increases the reporter ion signal intensity and improves the ratio accuracy, due to improved counting statistics, leading to a significant increase in the number of quantified peptides. Following MS2, SPS enables the simultaneous isolation of up to 20 MS2 fragment ions that undergo higher energy collisional dissociation (HCD) fragmentation with the MS3 fragments detected in the Orbitrap analyzer. This technique further minimizes co-ion interference and ratio compression compared to MS2.
Related resources
White Paper: High Throughput Quantitative Proteomics Using Isobaric Tags
Peer Reviewed article: Sensitive and Accurate Quantitation of Phosphopeptides Using TMT Isobaric Labeling Technique (Orbitrap Fusion Lumos)
Poster Note: Sensitive and accurate quantitation of phosphopeptides using TMT isobaric labeling technique (Orbitrap Fusion, Orbitrap Fusion Lumos)
Thermo Scientific TMT Real-Time Search for SPS MS3 multiplexing workflow with unique, intelligent acquisition delivers unrivalled quantitation for multiplexed proteome analysis that is the most accurate, precise and cost effective for functional biology studies, and it is exclusive to the Orbitrap Eclipse Tribrid MS.
The implementation of Real-Time Search for SPS enables significant improvements to specificity, accuracy and analytical performance for TMT experiments. This approach relies on searching MS/MS spectra in real-time against a user provided database and only performing SPS for identified spectra, thereby eliminating MS3 acquisition for unidentified spectra. This translates to increased sampling of precursors, resulting in 30-50% more identified peptides and increased accuracy in quantitation. Data indicates that the Real-Time Search for SPS MS3 workflow provides dramatic performance improvements for quantitative multiplexed experiments.
Free ebook: TMT is opening doors to a whole new view of biology with mass spectrometry
Advanced TMT quantitation workflows are making it possible to sensitively detect rare peptide species, to identify differences between healthy and diseased tissue, and conduct sophisticated surveys of the cellular proteome. This ebook provides an overview of TMT technology, and offers five case studies that illustrate some of the cutting-edge applications for which these reagents are being used.
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