MS-Based Protein-Protein Interaction Analysis Service

Over recent decades, proteomics driven by mass spectrometry (MS) has emerged as a critical methodology for analyzing protein-protein interactions (PPIs). Proteins exert their biological roles by forming macromolecular complexes through interactions with other proteins or molecules. Investigating these interactions provides deeper insights into protein functionalities. Techniques such as IP, Co-IP, or GST pull-down are employed to isolate target proteins, which are subsequently analyzed, identified, and quantified using MS. This characterizes the interacting proteins and further elucidates their functions. Affinity purification coupled with mass spectrometry (AP-MS) allows for the unbiased detection of interacting proteins under specific physiological conditions. One approach involves using the SILAC method to label cells in experimental and control groups, conduct Co-IP experiments based on antigen-antibody specificity to isolate immune complexes, and perform qualitative/quantitative analysis using LC-MS/MS. If a protein's levels in the experimental group are statistically different from those in the control group, it indicates an interaction with the protein under study, significantly reducing the likelihood of false positives in protein interactions. Combining SILAC and Co-IP-MS facilitates high-throughput quantitative analysis of protein interaction networks in specific contexts.

 

Services at MtoZ Biolabs

1. Protein Interaction Analysis Using Co-IP Combined with Mass Spectrometry

2. Protein Interaction Analysis Using GST Fusion Protein Pull-down Technology Combined with Mass Spectrometry

3. Protein Interaction Analysis Using SILAC Combined with IP Mass Spectrometry

 

Two common methods for mass spectrometry identification of interacting proteins are peptide fingerprinting and shot-gun proteomics. In peptide fingerprinting, protein samples are separated by SDS-PAGE. The polyacrylamide gel can be stained with coomassie brilliant blue or silver staining. However, it is necessary that the sample band is a single band, containing only one protein band. After cutting out the protein band, it is digested with enzymes, and analyzed by mass spectrometry. These digested peptides are then analyzed by mass spectrometry, and by comparing with a theoretical database, bioinformatics analysis assembles the information of the target protein. The limitation of peptide fingerprinting is that the method requires obtaining a single protein band during gel cutting; if there are impurity bands, they can affect the accuracy of detection, as the protein information in the impurity bands may mask the real target protein information. Therefore, it is more suitable for the identification of relatively pure protein samples. The strength of the shot-gun proteomics method lies in its ability to analyze protein mixtures.

 

The principle of shot-gun proteomics is to first digest the protein sample into peptides, then separate the peptides by high performance liquid chromatography (HPLC). The separated peptides are directly analyzed by a mass spectrometer. The mass spectrometry analysis includes two processes, primary mass spectrometry (MS1) and secondary mass spectrometry (MS2). Primary mass spectrometry captures the m/z and the intensity of peptides eluted in HPLC MS1; secondary mass spectrometry determines the amino acid sequence of peptides. MS2 selects individual peptides for fragmentation, thus obtaining the spectral data of that peptide. Search software matches the secondary mass spectrometry information with the corresponding database, combining match scoring and mismatch filtering to obtain the exact sequence of peptides, and then assembling the complete sequence of each protein, thereby identifying the protein.

 

MtoZ Biolabs can detect your protein samples after your protein interaction analysis experiment, or you can send samples to MtoZ Biolabs for a one-stop protein interaction detection service, including protein interaction experiments, IP, Co-IP, GST pull-down to protein sample mass spectrometry analysis. You only need to send your requirements and samples to us, and we will take care of all subsequent matters of the project, including sample pre-treatment, protein interaction experiments, mass spectrometry analysis, and analysis of original mass spectrometry data.