TMT labeling

TMT Based Proteomics ServiceProteomics techniques are evolving to become a highly sensitive, quantitative, andhigh-throughput approach to analyzing global protein dynamics within a cell, tissue oran organism. In the past decade, critical advances have been made across the entireproteomics workflow, most notably the development of Tandem Mass Tag (TMT) basedproteomics which has enabled more accurate, high-throughput quantitative proteomics.Therefore, Tandem mass tags (TMT) has become the widely used chemical labels formass spectrometry (MS)-based quantification and identification of biologicalmacromolecules such as proteins, peptides, and nucleic acids.As we known, proteins are the key functional actors in all cellular processes, anddiseases almost always manifest at the level of proteins. The analysis of all expressedproteins (the proteome), for example, healthy and diseased tissues, is definitely the mostapplicable approach to scientific areas, which are often named as biomarkers orfunctionally related protein signatures. As a result, a critical advanced Tandem MassTag (TMT) systems have been made across the entire proteomics workflow. In 2003,Thompson et al made the first attempt to analyze the simultaneous identification andrelative quantification of peptide pairs by Isobaric tags labeling technique. And thenTandem Mass Tag (TMT) system was designed by Thermo Fisher Scientific for theidentification and quantification of proteins in different kinds of samples.To be specific, Tandem Mass Tag (TMT) chemical labeling enables simultaneousprotein identification and relative quantitation of different samples (maximum is 10different samples) in the same proteomics experiment. TMT is classified as one of thereagents referred to as isobaric mass tags, which provide an alternative to gel- orantibody-based quantification but may also be used in combination with these and othermethods. Besides, each TMT tagging reagent is composed of an amine-reactive NHS-ester group, a spacer arm, and an MS/MS reporter. And the isobaric tagging reagentwithin a set has the same nominal parent (precursor) mass. These reporter ions are inthe low mass region of the MS/MS spectrum and are used to report expression changesin cell-based or tissue samples that may not be amenable to metabolic isotopic labelingstrategies such as SILAC. Isobaric chemical tags represent a fast, unbiased, andsensitive method to quantify proteins in a biological sample. They are used with avariety of samples including cells, tissues, and biological fluids, and enable relativequantitation simultaneously multiple conditions by mass spectrometry (MS).More specific, the isobaric labels TMT (tandem mass tagging) are available in up to 11tags that can be used for labeling practically any peptide or protein sample. TMT makesit possible to multiplex the analysis, to efficiently use the instrument time and exertfurther controls for technical variation. Thus, the wide application of TMT inquantitative protein biomarker discovery, this is because of its ability to multiplex upto 11 samples. TMTzero, TMTduplex, TMTsixplex, TMT10plex, and TMT11plex

Reagents share an identical structure, allowing efficient transition from methodsdevelopment to multiplex quantitation.TMT quantification is accomplished by measuring the intensities of fragment reporterions released from the labels in the tandem MS mode (MS2) during peptidefragmentation. Precursor ions are selected in the full scan mode (MS1) to be fragmentedthis is because the ion selection step reduces the noise levels, which is significantlyadvantageous. Ideally, only one selected precursor ion is fragmented during theprecursor ion fragmentation. However, it is common that other precursor ions arecaught within the specified m/z window and are fragmented together with the selectedprecursor in practice which is precursor co-isolation or mixing. Isobaric Labellingworks best with mass-spectrometers which allow MS3-level quantitation such asThermo’s Fusion Orbitraps. The additional filtering step allows almost completecorrection of co-isolation caused by contamination of MS1 precursors.One of the most important tasks of proteomics is measuring protein changes over timeor following stimuli. Several approaches have been developed for the relativequantification of proteins using mass spectrometry (MS). Isobaric labeling strategiesfor relative quantitative proteomics allow for parallel multiplexing of quantitativeexperiments. With the advancement of this technique, multiple peptide samples arechemically labeled with isobaric chemical tag variants and each variant has the samemolecular structure and mass. Each variant although is designed to produce a unique\"reporter ion\" when fragmented inside a mass spectrometer. Differentially labeledpeptides are indistinguishable in a first when peptide samples are labeled, combined,and analyzed using MS, MS spectrum of intact peptides. However, \"reporter ions\" canbe generated and recorded in a subsequent MS2 spectrum once each tag variant containsa labile component with the different mass. Intensities from each variant are recordedto represent the relative abundances of the peptide in each sample. When comes to thegeneral workflow of relative quantification of proteins, we normally use TMT by MS2or an additional MS3 spectrum.