High-throughput multiplexed quantitation
Tandem mass tags (TMT) are isobaric chemical tags that provide multiplexing capabilities for relative quantitative proteomics analysis. The ability to perform concurrent MS analysis of multiple samples increases throughput and enables relative quantitation of up to 10 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 quantition 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.
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. Protein identification is achieved by matching the resulting ion peaks to those provided in fragment databases. Comparing those ion peak intensities against the ones provided in the databases yields vital information for protein quantitation.
The Thermo Scientific TMT10plex Isobaric Label Reagent Set contains ten different isobaric compounds with the same mass and chemical (i.e., isotopomeric) structure. Each compound is composed of an amine-reactive NHS-ester group, a spacer arm, and a mass reporter. This reagent set can be used to prepare up to ten different peptide samples for parallel labeling and concurrent analysis. Increased multiplex relative quantitation and sample throughput, as well as fewer missed quantitative values, are just some of the advantages of using this approach, resulting in high-throughput quantitative proteomics.
The Thermo Scientific Orbitrap Fusion Tribrid Mass Spectrometer boasts the highest resolving power of all Orbitrap-based instruments (up to 500,000 @ m/z 200), achieving extreme detection limits and selectivity in full-scan MS mode. This system offers Synchronous Precursor Selection MS3 (SPS MS3) for highly accurate and precise TMT quantitation results. Such a unique acquisition strategy, exclusive to tribrid mass spectrometers, improves ratio accuracy for TMT reporter ions and boosts sensitivity, increasing total quantified peptides.
The Orbitrap Fusion Tribrid's segmented quadrupole improves ion transmission on narrow isolation windows, which is crucial for minimizing interferences. The brighter ion source permits 0.4 Th isolation windows and minimizes ratio inaccuracy.
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 qualtitative 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. It is also the only software that enables TMT-based glycopeptide quantitation.