The term "proteome" is derived from the combination of the words "protein" and "genome", meaning "the complete set of proteins expressed by a genome", that is, including all the proteins expressed by a cell or even a living organism. Proteomics essentially refers to the study of the characteristics of proteins at a large-scale level, including protein expression levels, post-translational modifications, protein-protein interactions, etc., thereby obtaining an overall and comprehensive understanding of processes such as disease occurrence and cellular metabolism at the protein level.
The research of proteomics can not only provide a material basis for the laws of life activities, but also offer theoretical basis and solutions for the clarification and conquest of the mechanisms of numerous diseases. Through comparative analysis of the proteome between normal individuals and pathological individuals, we can identify certain "disease-specific protein molecules", which can serve as molecular targets for new drug design or also provide molecular markers for the early diagnosis of diseases. Indeed, those drugs that sell well worldwide are themselves proteins or their target sites are certain protein molecules. Therefore, proteomics research is not only a necessary task for exploring the mysteries of life, but also can bring huge benefits to the cause of human health.
The development of proteomics technology has become an important support for the rapid development of modern biotechnology and has led to key breakthroughs in biotechnology. To help workers in the field of life sciences fully master the techniques and methods, experimental difficulties and key points, research frontiers and hotspots of proteomics, this technical platform will provide customers with proteomics technical services, including two-dimensional gel electrophoresis, isoelectric focusing, bio-mass spectrometry analysis and non-gel techniques.
Two-dimensional gel electrophoresis: The principle of two-dimensional gel electrophoresis is that in the first direction, isoelectric focusing is used to separate proteins based on their different isoelectric points, while in the second direction, SDS-PAGE is used to separate proteins based on their different molecular weights, thereby separating proteins in complex protein mixtures on a two-dimensional plane. Due to the significant position of two-dimensional electrophoresis technology in proteomics and medical research, it can be used in the study of protein transcription and post-transcriptional modification, comparison of proteomes and protein-protein interactions, research on cell differentiation and apoptosis, study of pathogenic mechanisms and drug resistance mechanisms, therapeutic effect monitoring, new drug development, cancer research, protein purity examination, and purification of small amounts of protein. The development of new alternative vaccines and many other aspects. In recent years, after multiple improvements, it has become the most valuable core method for studying the proteome.
Isoelectric focusing: Isoelectric focusing is an electrophoresis technique that emerged in the mid-1960s, which uses a medium with a pH gradient to separate proteins with different isoelectric points. Isoelectric focusing gel electrophoresis separates protein molecules based on their electrostatic charge or isoelectric point. In isoelectric focusing, protein molecules electrophoresis in a continuous and stable linear pH gradient formed by a carrier amphoteric electrolyte. The carrier amphoteric electrolyte is an aliphatic polyamino polycarboxylic acid, which forms a continuous pH gradient with the positive electrode being acidic and the negative electrode being alkaline in an electric field. Protein molecules carry electric charges under pH conditions that deviate from their isoelectric points, and thus can move in an electric field. When a protein migrates to its isoelectric point position, its electrostatic charge number becomes zero and it no longer moves in the electric field. Based on this, the protein is separated.
Biomass spectrometry: Biomass spectrometry technology is an important identification technique in proteomics research. Its basic principle is that after the sample molecules are ionized, the molecular weight is separated and determined based on the differences in the charge-to-mass ratio (M/E) among different ions. The target proteins separated by two-dimensional electrophoresis were enzymically hydrolyzed with trypsin (the peptide bonds formed by hydrolyzing the -C terminals of Lys or Arg) to form peptide segments, and these peptide segments were identified and analyzed by mass spectrometry. At present, the commonly used mass spectrometers include two types: 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 mode of MALDI was proposed by Karas and Hillenkamp in 1988. The fundamental principle of MALDI is to disperse the analyte in matrix molecules (nicotinic acid and its homologues) and form crystals. When the crystals are irradiated with a laser (a 337nm nitrogen laser), the matrix molecules absorb the laser energy, the sample is desorbed, and the charge transfer between the matrix and the sample causes the sample molecules to ionize. It generates ions from solid-phase specimens and determines their molecular weights in flight tubes. MALDI-TOF-MS is generally used for peptide mass fingerprinting, which is very fast (only 3 to 5 minutes per analysis), sensitive (reaching the fmol level), and can accurately measure the mass of peptide segments. However, if the peptide segments are not modified before analysis, MALDI-TOF-MS cannot provide the sequence of peptide fragments.
Electrospray mass spectrometry ESI-MS utilizes 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 the N2 gas flow, the solvent in the droplets evaporates, the surface area shrinks, and the surface charge density continuously increases until the generated Coulomb force and the surface tension of the droplets reach the Raili limit, and the droplets burst into charged sub-droplets. This process is repeated continuously, resulting in the final droplet being extremely fine and spray-like. At this point, the electric field on the droplet surface is very strong, ionizing the analyte and allowing it to enter the mass analyzer in the form of ions with single or multiple charges. ESI-MS generates ions from the liquid phase. Generally speaking, after the mixture of peptide segments is separated by liquid chromatography and analyzed by coupled and online connected ion trap mass spectrometry, the precise amino acid sequence of the peptide fragments is given. However, the analysis time is usually long.
At present, many laboratories combine two mass spectrometry methods to obtain peptide sequences of meaningful proteins and design probes or primers to acquire meaningful genes. With the in-depth research on proteomics, various new types of mass spectrometers have emerged, mainly based on the improvements and recombinations of the aforementioned mass spectrometers.
