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Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
MS is an analytical technique for the determination of the elemental composition of a sample or molecule. It allows rapid and high-through-put identification of proteins and sequencing of peptides most often after in-gel digestion. It is also used for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios. MS instruments consist of three modules: an ion source, which can convert gas phase sample molecules into ions (or, in the case of electrospray ionization (ESI), move ions that exist in solution into the gas phase); a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields; and a detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present. The technique has both qualitative and quantitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation. MS is now in common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds. MS is an important emerging method for the characterization of proteins. The two primary methods for ionization of whole proteins are ESI and matrix-assisted laser desorption/ionization. In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described earlier, and then introduced to a mass analyzer. This approach is referred to as “top-down” strategy of protein analysis. In the second, proteins are enzymatically digested into smaller peptides using proteases such as trypsin or pepsin, either in solution or in gel after electrophoretic separation. Other proteolytic agents are also used. The collection of peptide products are then introduced to the mass analyzer. When the characteristic pattern of peptides is used for the identification of the protein, the method is called peptide mass fingerprinting. If the identification is performed using the sequence data determined in tandem with MS analysis, it is called de novo sequencing. These procedures of protein analysis are also referred to as the “bottom-up” approach.
Comparative proteomic analysis revealed the metabolic mechanism of excessive exopolysaccharide synthesis by Bacillus mucilaginosus under CaCO3 addition
Published in Preparative Biochemistry & Biotechnology, 2019
Hongyu Xu, Zhiwen Zhang, Hui Li, Yujie Yan, Jinsong Shi, Zhenghong Xu
The stained gel images were captured using Image Master LabScan (GE Healthcare). Image data were analyzed using the PD-Quest 8.0.1 software (Bio-Rad).[21] Each sample was run in triplicate. Protein spots of interest were excised, and in-gel digestion with trypsin was performed as described.[22] Peptide mass spectra were obtained through MALDI TOF/TOF MS.
Hepatic proteomic assessment of oral ingestion of titanium dioxide nano fiber (TDNF) in Sprague Dawley rats
Published in Journal of Environmental Science and Health, Part A, 2022
Worlanyo E. Gato, Ji Wu, Isaac Appiah, Olivia Smith, Haresh Rochani
The gel bands were excised from the 1 D gel groups and shipped to the Proteomic and Mass Spectrometry Core Facility, University of Georgia, Athens GA for analysis. Then, gel bands were processed for in-gel trypsin digestion and peptide extraction using the following protocol: destaining, reduction and alkylation, in-gel digestion and extraction. The supernatant was analyzed directly without further processing.