TMT Phosphoproteomics
TMT phosphoproteomics is a high-throughput method based on TMT quantitative labeling technology, aimed at studying the dynamic changes in protein phosphorylation and their regulatory roles in cellular signaling, disease progression, and drug effects. As one of the post-translational modifications, protein phosphorylation is crucial in various biological processes, including cell cycle regulation, metabolic control, and immune response. By integrating enrichment strategies for phosphorylation and high-resolution mass spectrometry, TMT technology enables precise quantitative analysis of phosphorylated proteins across multiple samples, facilitating the discovery of key regulatory networks. This method is widely applied in the research of cancer, neurodegenerative diseases, cardiovascular diseases, and drug development, offering insights into disease mechanisms and potential therapeutic targets. TMT phosphoproteomics is distinguished by its high throughput, precision, and data reproducibility. Unlike traditional methods, TMT technology allows for the concurrent analysis of multiple samples in a single experiment, enhancing efficiency and minimizing batch effects. The peptide-level TMT labeling ensures data stability and reliability. However, challenges exist, such as co-elution interference, which may affect quantification accuracy, particularly in phosphorylation detection due to the phosphate group's instability, potentially leading to signal loss in some peptides.
The experimental procedure of TMT phosphoproteomics involves several key steps: sample preparation, TMT labeling, phosphopeptide enrichment, liquid chromatography separation, mass spectrometric analysis, and data processing. Initially, total proteins are extracted from tissues, cells, or biofluids and digested into peptides using enzymes like trypsin. These peptides are then chemically labeled with TMT tags specific to different experimental groups, allowing their origin to be distinguished in subsequent mass spectrometry. Given the typically low abundance of phosphorylation modifications, specific enrichment strategies such as Immobilized Metal Affinity Chromatography (IMAC) and titanium dioxide (TiO₂) microsphere techniques are employed to enhance detection sensitivity. The enriched phosphopeptides are then fractionated using high-performance liquid chromatography (HPLC) to reduce sample complexity before undergoing detailed analysis via high-resolution mass spectrometers like Orbitrap or Q-TOF. During the MS2 or MS3 stages, TMT tags release distinctive reporter ion signals, enabling the calculation of phosphopeptide relative abundances across samples to gather comprehensive quantitative data on phosphorylated proteins. Bioinformatics tools such as Proteome Discoverer and MaxQuant are subsequently used for protein identification, phosphorylation site analysis, and functional enrichment, uncovering potential signaling pathways and regulatory mechanisms.
Data analysis in TMT phosphoproteomics includes critical steps such as identifying phosphorylated proteins, calculating quantities, analyzing phosphorylation differences, and constructing enriched pathways and networks. Researchers employ database algorithms like Sequest and Mascot to identify phosphorylated proteins and their modification sites, using statistical methods to identify phosphopeptides with significant differences between experimental groups. These sites often correspond to crucial nodes in signaling pathways like MAPK, PI3K-Akt, and JAK-STAT, which are vital for processes such as cell proliferation, differentiation, and metabolism. Furthermore, databases like KEGG and GO are used for functional enrichment analysis to elucidate the roles of phosphorylated proteins in specific biological contexts.
As technology advances, TMT phosphoproteomics is evolving towards greater sensitivity, broader coverage, and reduced background noise. Innovations in enrichment methods, such as MOAC-Ti4+ and polydopamine-coated magnetic microspheres, have notably improved the detection efficiency of low-abundance phosphopeptides. MtoZ Biolabs leverages state-of-the-art mass spectrometry and extensive expertise in proteomics to provide high-quality phosphoproteomics analysis services.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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