Post-translational Modification Analysis

1. Post-translational Modification Analysis Protein Post-Translational Modification (PTM) plays a very important role in living organisms and is closely related to gene expression, signal transduction pathways, cell division and other processes. The modification of the same protein in different physiological states, different subcellular localization and different amino acid sites may correspond to different functions. Therefore, posttranslational modification of proteins is a hot and difficult problem. Protein post-translational modification is a qualitative analysis of purified or complex protein samples. The common types of modification include phosphorylation, methylation, acetylation, biotinization, phosphorylation, glycosylation, ubiquitination, disulfide, etc. 1.Theory: Protein amino acid sequence of a specific location can covalently bind with chemical group or small molecular weight of protein so that to get protein post-translational modifications (PTMs). Compared to proteins that have not been modified, PTMs can lead to an increase in the molecular weight of specific sequences, and specific post-translational modifications can be detected by high-precision mass spectrometry detection. Mass spectra can distinguish the changes of molecular weight before and after modification of protein, so as long as the precise changes of molecular weight before and after modification of target protein can be known, any post-translation modification methods can be identified and quantified. 2. Technical feature:

2. 1) High sensitivity, minimum protein concentration can be fmol level. 2) Protein that does not require special purification. 3) There is no risk of radioactivity compared to conventional methods. 4) Flux is high. 5) High accuracy. Here are some common ways for protein post-translational modification: Protein Phosphorylation Analysis Phosphorylation has the characteristics of simplicity, flexibility and reversibility, as well as the availability of donor ATP of phosphate groups. It is a common regulation method of eukaryotic cells. The reversible process of phosphorylation and dephosphorylation of proteins, almost regulates all the process of life activity including cell proliferation, development, differentiation, regulation and control of the cytoskeleton, cell apoptosis, neural activity, muscle contraction, metabolism etc. And reversible protein phosphorylation is the most important signal transduction mode known. Many human diseases are known to be caused by abnormal phosphorylation, and some phosphorylation is caused by a disease. Protein N-acetylation Analysis N-acetylation is a highly conserved and reversible protein modification in vivo, which plays an important role in the activation of transcription regulatory factors in the nucleus. In addition, a large amount of non-histone

3. acetylation is involved in the regulation of metabolic pathways and the activity of metabolic enzymes. Because of the low content and wide dynamic range of acetylated modified proteins in biological samples, it is necessary to enrich the acetylated peptide fragments to improve their abundance before mass spectrometry analysis. Then, traditional quantitative proteome analysis method was used to quantitatively analyze the concentrated acetylated peptide sample. Protein Ubiquitination Analysis

4. Ubiquitination is an important posttranslational modification. The ubiquitin-proteasome system mediates 80%~85% protein degradation in eukaryotes. What’s more, ubiquitination can directly affect the activity and localization of proteins and regulate a variety of cellular activities including cell cycle, apoptosis, transcriptional regulation, DNA damage repair and immune response. Ubiquitination is closely related to the incidence of diseases such as tumor and cardiovascular diseases. Therefore, as a major achievement of biochemical research in recent years, it has become a new target for research and development of new drugs. Protein Glycosylation Glycosylation is a process of adding saccharides to proteins or lipids under the control of enzymes, occurring in areas such as endoplasmic reticulum and Golgi bodies. Under the action of glycosyltransferase, the saccharides are transferred to the protein and covalently binds to the amino acid residues on the protein. Proteins undergo glycosylation to form glycoproteins. Glycosylation is an important modification of proteins, and has the function of regulating proteins. Glycosylation not only affects the spatial conformation, biological activity, transport and localization of proteins, but also plays an important role in molecular recognition, cell communication, signal transduction and other specific biological processes.

5. It can be said that from medical health to agricultural production, protein is closely related to people's life. However, there are still many aspects of post-translational modification that are not yet understood and many that have not yet been discovered. This makes the research on post-translational modification of proteins a hot spot in the field of life science.