The study of the affinity between disease-related drug target proteins and small molecular compounds is a hot field in the development of new drug molecules. Affinity Purification Mass Spectrometry , as a method for indirect screening of small molecule ligands, has been successfully applied to the screening of ligands of many receptors, enzymes and other target proteins, and has been widely used.
AP-MS is a unique platform for the discovery of novel PPIs that can be applied to large-scale analysis. Using affinity tags, AP-MS separates interacting proteins bound to target proteins and induces cleavage of these proteins into polypeptides by protease treatment. The resulting peptides are ionized and then separated according to their mass-to-charge ratio (m/z). The sensitivity of mass spectrometry-based proteomics is further enhanced by the entire liquid chromatography and tandem mass spectrometry analysis, which enables the detection of post-translational modifications even at single residues. AP-MS is a database-dependent analysis that provides a good amount of data for the identification of PPIs in a single sample.
The basic principle of affinity mass spectrometry is to first obtain a complex of ligands bound to a target protein, then use a purification method to separate the complex from the unbound ligands in solution, and finally perform liquid-mass spectrometry on the ligands dissociated from the complex to identify their ligand structures. Researchers usually select small molecule ligands that specifically bind to target proteins from a large library of compounds, and such a mixture screening approach can significantly improve screening throughput and reduce costs.
1. The interacting protein obtained by AP-MS is bound to the bait protein in the cell, which conforms to the real physiological situation in vivo, and the obtained results are highly reliable.
2. Unlike CO-IP, which adopts antibodies to pull protein complexes, it can effectively avoid antibody contamination.
3. The obtained eluate can be directly enzymatically hydrolyzed and analyzed by mass spectrometry, which greatly reduces the protein loss caused by gel running.
Creative Proteomics has a complete molecular biology, cell biology, protein immunology and mass spectrometry platform, specializing in protein-protein, protein-nucleic acid and nucleic acid-nucleic acid interaction screening and validation, and are committed to providing high-quality technical services for biopharmaceutical companies, universities, research institutions, hospitals and researchers.
1. Construction of target gene expression vectors containing SBP tags.
2. Overexpression of tagged decoy proteins. Western blot (WB) detection of cell lysates (transient transfection is not limited by gene size, but is influenced by the rate of cell transfection, so hard-to-transfect cells may not be suitable for this method and require other gene introduction methods, such as lentivirus or adenovirus).
3. Purification and elution of the protein complexes with strep magnetic beads.
4.The eluates from experimental and control groups were digested by trypsin separately to obtain 2 groups of peptide mixtures.
5. LC-MS/MS analysis of the 2 groups of peptide mixtures to obtain qualitative information of the proteins.
6. Analytical results and report.
Approaches to q-AP-MS experiments
The name, sequence, species and other related information of the bait gene or the cDNA template of the bait gene, etc.
Cells to be studied (ensure that the cells are in good condition and are easy to be transfected with plasmids) and their culture information.
The constructed eukaryotic expression vector
The identification results of mass spectrometry of the experimental and control proteins
Results of data analysis