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Techniques for Quantitative Proteomics

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With the development and maturity of mass spectrometry, the technical advantages of high throughput, high sensitivity and wider dynamic range have been emerged, which provides a reliable research method for proteomics research. In recent years, quantitative proteomics has become one of the research hotspots in omics field. Quantitative proteomics  is an emerging technology used for accurate quantification and identification of proteins expression in biological samples. It can not only identify the proteins expressed in different states, but also accurately quantify their abundance. According to different quantitative purposes, quantitative proteomics can be divided into two categories relative quantitative proteomics and absolute quantitative proteomics. Relative quantitative proteomics, also known as comparative proteomics, is a comparative analysis of protein expression level in cells, tissues or organism in different physiological or pathological conditions. Absolute quantitative pr
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Practice of N-terminal Sequencing by Edman Degradation Technology in Protein and Peptide Sequence Analysis

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The N-terminal amino acid sequence of protein is one of the key quality attributes of biological drugs. N-terminal sequencing by Edman degradation  is a routine method to analyze this property. The analysis of 15 amino acid sequences on the N-terminal by Edman degradation method is usually a necessary item when applying for biological drugs . At  the same time, it is also an annual inspection item of many listed biological drugs (see Pharmacopoeia 2015). In scientific research, N-terminal sequencing can be used to provide key information for the confirmation of unknown or uncertain theoretical sequences. So this method is widely used in scientific research and industry.  W hat are its principles ? H ow to analyze the results ? Advice for sample delivery? P rinciples In short, we can understand it from these two aspects: The first step  is Edman degradation . T hrough the reaction of PITC (Phenylisothiocyanate), an organic reagent, and α - amino acid at the N-terminal of pr
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De Novo Protein Sequencing Procedures and Features

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The sequence of a protein molecule is the basis for the function of a protein. Accurate determinat ion of protein sequence plays an important role in the study of protein functional epitopes, detection of commercial monoclonal antibodies, vaccines, and kits. Although there are many studies on protein sequencing, accurate sequencing of proteins is still not as fast and accurate as that of DNA. Protein sequencing is mainly hampered by the purification of protein samples, and the complexity of factors that make up the amino acid com position of proteins. De novo   protein sequencing  is  also called novel protein sequencing. This technique deduces t he amino acid sequence based on the difference in mass between a series of regular fragment ions generated by the peptide collision with an inert gas. We can guess the amino acid sequence and post-translational modifications based on the y ion and b ion at the cleavage of the peptide bond. De novo  sequencing has a unique advantage that t
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Protein Data-independent Acquisition Quantitative Technology

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In the field of proteomics research, liquid chromatography-mass spectrometry (LC-MS/MS)-based discovery proteomics can detect and relatively quantify thousands of proteins in biological samples over the past decade or two. It has been widely used in various research, and reseaechers have developed two mainstream methods, label quantification (iTRAQ/TMT, SILAC) and label free, which are based on data-dependent acquisition (DADA). The method of collecting protein spectral data is also referred to as "shotgun". However, the DDA data acquisition mode has inherent shortcomings such as poor repeatability, inaccurate quantification, large data loss, and difficulty in detecting low abundance proteins. MRM /SRM/PRM-based target proteomics can provide accurate absolute quantitative results for tens to hundreds of target proteins with high repeatability and is the gold standard for absolute quantitation of mass spectrometry. However, the flux is relatively low, and accurate quantitati
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Effects of Glycosylation on the Stability and Half-life of Antibody Drugs

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Read   Effects of Glycosylation on the Stability and Half-life of Antibody Drugs Effects of Glycosylation on the Safety of Antibody Drugs The Critical Quality Attributes   (CQA) of antibody is the standard to judge whether it is qualified. CQA   must be in a proper range to ensure the effectiveness of drugs, and more importantly, the safety of drugs. By affecting the effect function of F c  end, glycosylation modification of F c  end will affect the safety of antibody, which is manifested in immunogenicity, PK/Pd (pharmacokinetics/pharmacodynamics), etc. The development of antibody has experienced mouse antibody, chimeric antibody, humanized antibody, and then to the last all human antibody, throughout which the immunogenicity of antibody has been constantly reduced. For glycosylation , human cells specifically synthesize Neu5Ac sialic acid, while other mammalian cells can not only synthesize Neu5Ac sialic acid, but also synthesize Neu5Gc sialic acid with immunogenicity
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Effects of Glycosylation on the Stability and Half-life of Antibody Drugs

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Among various post-translational modifications of proteins, glycosylation is one of the most important and complex modifications, and it is also one of the key quality attributes to evaluate antibodies. The realization of the function of monoclonal antibody is closely related to its glycosylation, which will affect the performance of protein, such as conformation, stability, solubility, pharmacokinetics, activity and immunogenicity. Glycosylation is one of the important post-translational modifications of proteins. According to the modified sites of glycosylation, it can be divided into N-glycosylation and O-glycosylation. N-glycosylation is located in asn-297. N-acetylglucosamine in oligosaccharide links itself with  amide nitrogen on asparagine residue  to  modify   protein ,which start s  from endoplasmic reticulum and complete s  in Golgi body .  O-glycosylation is completed in Golgi body by n-acetylgalactose in oligosaccharide linking with hydroxyl on serine or threonine re
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