Phospho Proteomics Mass Spectrometry
Phospho proteomics mass spectrometry is an efficient method for investigating the dynamic changes in protein phosphorylation modifications within cells. As a post-translational modification, protein phosphorylation predominantly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues. It plays a crucial role in regulating protein activity, stability, subcellular localization, and interactions, thereby influencing nearly all biological processes. Abnormal phosphorylation is linked to a range of diseases, including cancer, neurodegenerative disorders, and metabolic diseases. Consequently, phospho proteomics mass spectrometry is invaluable for elucidating the distribution and regulatory mechanisms of phosphorylation modifications, which is essential for both basic research and clinical applications. Despite the significant progress in this domain, practical application still faces some challenges. Phosphorylated peptides are typically low in abundance and prone to degradation, necessitating efficient enrichment and stable processing workflows. Additionally, mass spectrometry's detection sensitivity limits the identification of low-abundance phosphorylation sites. Moreover, analyzing the interactions of post-translational modifications, such as between phosphorylation and acetylation, requires further methodological development. With advancements in mass spectrometry technology and bioinformatics, the resolution and coverage of phospho proteomics mass spectrometry are expected to improve further. The use of ultrahigh-resolution mass spectrometers, like Orbitrap and TOF instruments, enhances the analysis of complex phosphorylation patterns. Additionally, integrating multi-omics data, such as genomics and metabolomics, provides a comprehensive understanding of phosphorylation's role in biological systems.
Principles and Process of Phospho Proteomics Mass Spectrometry Analysis
Phospho proteomics mass spectrometry integrates high-resolution mass spectrometry with advanced sample preparation techniques, enabling high-throughput analysis of phosphorylation sites and their dynamic changes in complex samples. The typical workflow includes protein extraction, enzymatic digestion, enrichment of phosphorylated peptides, mass spectrometric analysis, and data interpretation.
1. Protein Extraction and Enzymatic Digestion
Proteins are extracted from cells or tissues and digested into shorter peptides using enzymes such as trypsin. Due to the low abundance of phosphorylated peptides and the presence of numerous non-phosphorylated peptides, subsequent enrichment is necessary.
2. Enrichment of Phosphorylated Peptides
Enrichment is critical in phospho proteomics mass spectrometry. Common methods include metal oxide affinity chromatography (MOAC), immunoaffinity enrichment (IMAC), and titanium dioxide (TiO₂) microspheres. These techniques selectively bind phosphate groups, enhancing the signal-to-noise ratio of phosphorylated peptides in mass spectrometry.
3. Mass Spectrometry Analysis
Enriched samples are analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liquid chromatography separates complex peptides, and tandem mass spectrometry, through collision-induced dissociation (CID) or high-energy collision dissociation (HCD), generates fragment ion spectra to deduce peptide sequences and phosphorylation sites.
4. Data Interpretation and Bioinformatics Analysis
Software tools, such as MaxQuant or Proteome Discoverer, interpret mass spectrometry data, identifying phosphorylation sites and quantifying their variations across samples. Further bioinformatics analysis uncovers the role of phosphorylation modifications in signaling pathways and cellular networks.
Applications of Phospho Proteomics Mass Spectrometry Analysis
1. Study of Cellular Signaling Pathways
Phosphorylation is a central regulatory mechanism in many signaling pathways. Mass spectrometry allows researchers to track changes in protein phosphorylation induced by stimuli or drug treatments, facilitating the construction of signaling networks. For example, understanding the dynamic phosphorylation changes in ERK and AKT pathways is crucial in cancer, immune response, and cell cycle regulation.
2. Disease Mechanism Analysis
Diseases such as cancer and diabetes are often associated with abnormal protein phosphorylation. Phospho proteomics mass spectrometry can identify disease-related phosphorylation abnormalities, aiding in the discovery of potential biomarkers and drug targets.
3. Drug Development
In drug development, phospho proteomics analysis is instrumental in designing targeted therapies. By elucidating the phosphorylation patterns and regulatory mechanisms of target proteins, more precise drugs can be developed. Moreover, examining the phosphoproteome's response to drug treatment can evaluate drug mechanisms and efficacy.
4. Basic Biological Research
Phospho proteomics mass spectrometry is a powerful tool for exploring fundamental biological processes like cell cycle, metabolic regulation, and transcriptional control. For instance, phosphorylation dynamics during mitosis reveal mechanisms of key regulatory proteins.
MtoZ Biolabs boasts a robust technological system and extensive expertise in phospho proteomics. We offer comprehensive services, from sample processing and phosphorylated peptide enrichment to high-resolution mass spectrometry, accurately identifying phosphorylation sites and elucidating their functional significance.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
