Overview of Single/Mixed Protein Separation and Purification Methods and Mass Spectrometry Identification

In terms of common identification method of a single protein, you may immediately think of Western Blot. Western Blot identifies a target protein by specific binding of the antibody to the target molecule, but this method is suitable for identifying known proteins and must be applied to antibodies, and is not suitable for proteins with unknown or without corresponding antibodies. What if the protein to be studied happens to be unknown or has no antibody available? In this case, the protein can be identified by means of mass spectrometry.

Single Protein Separation and Purification

When it comes to single protein sample, as the name suggests, this type of protein sample should have undergone a certain protein purification step to obtain a relatively single protein. Generally, proteins synthesized by cell lysis or in vitro contain other heteroproteins, so it is necessary to distinguish the target protein from the heteroprotein. The commonly used protein separation and purification method is electrophoretic separation. One-dimensional electrophoresis separation utilizes different protein size characteristics, and can distinguish proteins of different molecular weight well. In addition, two-dimensional electrophoresis can further distinguish protein molecules with different molecular weights.

One-dimensional Electrophoresis

Currently, the widely used one-dimensional electrophoresis is vertical plate polyacrylamide gel electrophoresis (SDS-PAGE). The principle is that different concentrations of vinyl amide will produce a gel with a certain pore size after polymerization, and then add to the buffer. The SDS surfactant neutralizes the charge on the surface of the protein. When the denatured protein mixed sample passes through the gel gap, proteins of different molecular weights migrate in the gel at different rates and are thus separated. One-dimensional electrophoresis is capable of separating the initial separation of most protein and protein complexes.

Two-dimensional Electrophoresis

Two-dimensional electrophoresis, also known as dimensional electrophoresis, adds lateral isoelectric focusing separation to one-dimensional electrophoresis, and its longitudinal separation is identical to that of one-dimensional SDS separation. Two-dimensional electrophoresis firstly uses the different characteristics of the isoelectric point of the protein to separate the protein, and then further separates the proteins with the same or similar isoelectric points according to the molecular weight of the protein, further improving the protein separation efficiency. The protein resolution of two-dimensional electrophoresis has been greatly improved compared with that of one-dimensional electrophoresis. After two-dimensional electrophoresis separation, most proteins can be separated into single protein spots.

Single Protein Identification

Current methods for the identification of higher purity or single proteins are protein fingerprinting (PMF) and MS/MS tandem mass spectrometry. In both methods, the protein is first cut into polypeptide fragments, and then the peptide is analyzed by a mass spectrometer to achieve the purpose of protein identification. The difference between the two methods is that the PMF method only uses the first-stage mass spectrometry to detect the protein peptides, while the MS/MS not only detects the first-stage mass spectrometry, but also selects specific peptides for peptide fragmentation and peptide sequence determination. Therefore, MS/MS is more accurate than PMF, and has higher reliability than database comparison, but it is more complicated to analyze.

Protein Fingerprint Identification (PMF)

Due to the difference in protein sequence, the size of the polypeptide fragments produced by digestion of different proteins under the same digestive enzyme is specific. Protein fingerprinting identification method utilizes the different characteristics of each fragment produced by each protease to digest the purified protein, and then analyze the mass of the peptide fragment produced by digestion to obtain the peptide mass spectrum. The peptide spectrum is then compared with the protein theoretical peptide spectrum in the database, and the identity information of the protein can be obtained.

MS/MS Tandem Mass Spectrometry

The principle of MS/MS detection of protein is to firstly detect the mass information of the peptide using the first-order mass spectrometer, and then select the peptide with high peak to be crushed and analyze to obtain the secondary mass spectrum. The search software is used to select the corresponding database to analyze the mass spectrometry data, and the identification result is judged in the form of scoring. When the score is greater than a certain threshold, it indicates that the mass spectrometry identification is successful, and vice versa.

Protein Mixture Identification

Although there are many methods for protein separation and purification, in many cases the separation efficiency is not high enough, and the isolated protein may still contain multiple proteins. In addition, proteins with lower abundance may cause further loss during the separation process and cannot be identified. Therefore, it is necessary to directly analyze this type of protein without isolation. Current method for the analysis of mixed proteins is LC-MS/MS chromatography mass spectrometry. Compared with the PMF method and the MS/MS method, liquid chromatography separation is added before LC-MS/MS mass spectrometry identification, which makes the protein analysis more accurate and can achieve 100% accuracy for peptide sequence determination. Therefore, this service can identify more proteins than single protein tape identification. LC-MS/MS can be used to identify protein mixtures samples of up to 200 proteins, such as immunoprecipitated samples, Pull-down samples, and so son.

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