Over the past few decades, mass spectrometry (MS)-based proteomics has become a crucial technology for identifying protein-protein interaction analyses (PPIs). Proteins exert their biological functions by interacting with other proteins or compounds to form macromolecular complexes. Studying protein interactions can help us better understand proteins. After obtaining the target proteins through interaction analysis methods such as IP, Co-IP, or GST pull-down, mass spectrometry is used to analyze, identify, and quantify the proteins to characterize interacting proteins and further analyze protein functions. Affinity purification combined with mass spectrometry (AP-MS) allows unbiased detection of interacting proteins under specific physiological conditions. One approach is to use the SILAC method to label experimental and control group cells separately, then perform a Co-IP experiment to isolate the immune complex through specific antigen-antibody reactions; followed by LC-MS/MS to qualitatively/quantitatively detect proteins in the immune complex. When there is a statistically significant difference in the amount of a protein between the experimental and control groups, it is determined that this protein interacts with the protein under study, greatly reducing the possibility of false-positive results in protein interactions. The combination of SILAC technology and Co-IP-MS technology can be used for high-throughput quantitative analysis of protein interaction networks under specific conditions.
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Mass spectrometry analysis of protein interactions
Two common methods for identifying interacting proteins via mass spectrometry are peptide fingerprinting and shotgun proteomics. Peptide fingerprinting involves separating protein samples using SDS-PAGE. The polyacrylamide gel can be stained with Coomassie Brilliant Blue or silver stain. However, the sample band must be a single band containing only one protein. After enzymatically digesting the excised protein band, mass spectrometry is used for analysis. The resulting peptides are analyzed by mass spectrometry, and bioinformatics analysis compares them with theoretical databases to reconstruct the target protein information. A limitation of peptide fingerprinting is that it requires obtaining a single protein band during gel cutting, and contamination can affect detection accuracy by obscuring the true target protein information. Therefore, it is more suitable for identifying relatively pure protein samples. The strength of shotgun proteomics is its ability to analyze protein mixtures.
The principle of shotgun proteomics is to first digest protein samples into peptides, then separate the peptides using high-performance liquid chromatography (HPLC). The separated peptides are directly analyzed by mass spectrometry. Mass spectrometry analysis includes two processes: MS1 and MS2. MS1 captures the mass-to-charge ratio (m/z) and intensity of peptides eluted in HPLC MS1; MS2 selects individual peptides for fragmentation to obtain spectral data. Software searches compare MS2 information with corresponding databases, using match scores and mismatch filtering to determine the exact peptide sequences, thereby reconstructing complete protein sequences and identifying proteins.
Biotech company BGI can detect protein samples after protein interaction analysis experiments or accept samples you send, offering one-stop interaction protein detection services including protein interaction experiments, IP, Co-IP, GST pull-down, and mass spectrometry analysis. Simply send us your requirements and samples, and we will handle all subsequent project tasks, including sample pretreatment, protein interaction experiments, mass spectrometry analysis, and analysis of raw mass spectrometry data.
