Mass Spectrometry Analysis Expert provides the latest proteomics, metabolomics information based on years of experience in this field.
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 quantitation of high-throughput proteins cannot be performed.
Is there a technology, which is not only of high-throughput, quantitative,but with accurate repeatability as well? The DIA technology (data-independent acquisition) developed and rapidly ignited in recent years is such the technology.
This technology combines the high-throughput and mass spectrometry of the traditional proteome shotgun with the quantitative and accurate advantages of the absolute quantitative gold standard MRM/PRM, which is named the most noteworthy technology of 2015 by Nature Methods. The number of published articles related to DIA has grown rapidly in the past three years and is the current fragrance in the field of proteomics research.
Protein DIA Quantitative Publication Trend (data source: Pubmed) DIA: DIA/SWATH The entire scanning range of the mass spectrum is divided into several parts, and all the parent ions in each part are selected, fragmented, and detected at high speed and cyclically, and all pieces of information of all the parent ions in the sample are obtained without any deviation.
Protein DIA Quantitative Analysis Workflow Applications of Protein DIA Quantitative Analysis: Medicine, botany, pharmacy, animal husbandry, food and environment.
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
After the protein is translated in the cell, it is processed through a very important step before being transported to the corresponding organelle and undergoing specific biological effects. The role of protein post-translational modification ( PTM ) is primarily to alter the activity, localization or function of the protein. Protein PTM also further increases the diversity and complexity of cellular pathway mechanisms and life activities. Except for acetylation , c ommon protein PTM include s phosphorylation , glycosylation , ubiquitination , and the like. Protein A cetylation M odification Protein acetylation modification, as the name suggests, refers to the grafting of acetylated groups on the original basis of the protein. In cells, the acetylation modification reaction is catalyzed by an acetyltransferase, and the acetyl group of acetyl-CoA is transferred and added to the protein lysine residue. In earlier studies, acetylation has been considered to be a pos
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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