Techniques and Methods of Proteomics


mass spectrometry

The development of proteomics technology has become an important support for the rapid development of modern biotechnology, and will lead to key breakthroughs in biotechnology. In order to help workers in the field of life sciences to fully grasp the technology and methods of proteomics, experimental difficulties and key points, research frontiers and hotspots, this technology platform will provide customers with proteomics technical services, including two-dimensional gel electrophoresis, isoelectric Focusing, biological mass spectrometry and non-gel techniques.

Two-dimensional gel electrophoresis

The principle of two-dimensional gel electrophoresis is that the first dimension is separated by isoelectric focusing based on the isoelectric point of the protein, and the second dimension is separated by SDS-PAGE according to the difference in molecular weight, and the proteins in the complex protein mixture are separated on a two-dimensional plane. . Due to the important position of two-dimensional electrophoresis in proteome and medical research, it can be used for protein transcription and post-transcriptional modification studies, proteome comparison and protein-protein interactions, cell differentiation and apoptosis studies, pathogenic mechanisms and resistance Drug mechanism research, efficacy monitoring, new drug development, cancer research, protein purity inspection, small protein purification, development of new alternative vaccines and many other aspects. In recent years, it has become the most valuable core method for studying proteome after many improvements.

Isoelectric focusing

Isoelectric focusing (IEF) is an electrophoresis technique developed in the mid-1960s that uses a medium with a pH gradient to separate proteins with different isoelectric points. Isoelectric focusing gel electrophoresis separates protein molecules according to their electrostatic charge or isoelectric point. In isoelectric focusing, protein molecules are electrophoresed in a continuous and stable linear pH gradient formed by a carrier ampholyte. The carrier ampholyte is an aliphatic polyamino polycarboxylic acid, which forms a continuous pH gradient in which the positive electrode is acidic and the negative electrode is basic in an electric field. Protein molecules are charged under pH conditions that deviate from their isoelectric point, so they can move in the electric field; when the protein migrates to its isoelectric point, its electrostatic charge is zero and no longer moves in the electric field, according to which protein isolation.

Biological mass spectrometry

Biological mass spectrometry is the most important identification technology in proteomics research. The target protein separated by two-dimensional electrophoresis is digested with trypsin (the peptide bond formed by the hydrolysis of the -C-terminal of Lys or Arg) into peptide fragments, and these peptide fragments are identified and analyzed by mass spectrometry. Two types of mass spectrometry are commonly used today: matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray mass spectrometry (ESI-MS).

Time-of-flight mass spectrometry

The ionization method of MALDI was proposed by Karas and Hillenkamp in 1988. The basic principle of MALDI is to disperse the analyte in matrix molecules (nicotinic acid and its homologues) and form crystals, when the crystal is irradiated with a laser (nitrogen laser at 337 nm), the matrix molecules absorb the laser energy, the sample desorbs, the matrix- Charge transfer occurs between the samples to ionize the sample molecules. It generates ions from solid-phase samples and determines their molecular weight in a flight tube. MALDI-TOF-MS is generally used for peptide mass fingerprinting, which is very fast (only 3~5min per analysis) and sensitive (up to fmol level) , the peptide mass can be accurately measured, but MALDI-TOF-MS cannot give the sequence of the peptide fragment if the peptide is not modified before analysis.

Electrospray mass spectrometry

ESI-MS uses a high electric field to charge the droplets flowing out of the capillary column at the injection end of the mass spectrometer. Under the action of N2 gas flow, the droplet solvent evaporates, the surface area is reduced, and the surface charge density increases continuously until the generated Coulomb force and the droplet surface. The tension reaches the Rayleigh limit, and the droplet bursts into charged sub-droplets. This process is repeated to make the final droplet very fine and spray-like. At this time, the electric field on the surface of the droplet is very strong, ionizing the analyte and carrying The singly or multiply charged ions enter the mass analyzer. ESI-MS generates ions from the liquid phase. Generally speaking, after the mixture of peptide fragments is separated by liquid chromatography, it is analyzed by coupled and on-line ion trap mass spectrometry to give the precise amino acid sequence of the peptide fragment, but the analysis The time is generally longer.
At present, many laboratories combine the two mass spectrometry methods to obtain the peptide sequences of meaningful proteins, and design probes or primers to obtain meaningful genes. With the deepening of proteome research, a variety of new mass spectrometers have appeared, mainly based on the above-mentioned mass spectrometers for improvement and recombination.