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Proteins and Proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
Shotgun proteomics is a method of identifying proteins in complex mixtures using a combination of high-performance liquid chromatography combined with mass spectrometry. The name is derived from shotgun sequencing of DNA, which is itself named by analogy with the rapidly expanding, quasi-random firing pattern of a shotgun. In shotgun proteomics, the proteins in the mixture are digested and the resulting peptides are separated by liquid chromatography. Tandem mass spectrometry is then used to identify the peptides.
Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Shotgun proteomic is a method of identifying proteins in complex mixtures using a combination of HPLC and MS. The name is derived from shotgun sequencing of DNA, which is itself named by analogy with the rapidly expanding, quasi-random firing pattern of a shotgun. In shotgun proteomics, the proteins in the mixture are digested, and the resulting peptides are separated by liquid chromatography. Tandem MS is then used to identify the peptides.
Sludge: next paradigm for enzyme extraction and energy generation
Published in Preparative Biochemistry and Biotechnology, 2019
Santosh Kumar Karn, Awanish Kumar
Humic acids contamination in protein could interfere with the visualization of protein bands in SDS-PAGE.[50] In addition, for some special proteins, such as having extremes in isoelectric points, molecular mass, and hydrophobic nature, the two-dimensional gel electrophoresis/MS technique is often difficult to distinguish. Fortunately, shotgun proteomics is an indirect method of measurement amino acid sequences of peptides after proteolytic digestion of whole proteins.[51] Due to merit and qualities more easily fragmented, fractionated, ionized, shotgun proteomics is more broadly applied for protein analysis.[52] Therefore, this method is much suitable for determining the EPS with low-protein concentration and many disruptors. Researchers are making efforts towards extracting metaproteome, taking into account to get the highest number of protein identifications, Zhang et al.[53] extracted extracellular proteins in wastewater treatment plant sludges and identified using shotgun proteomics, from anaerobic, anoxic and aerobic samples and extracted 130, 108, and 114 proteins, respectively. Most of the proteins originated from the cell and cell part and their molecular functions binding activity, catalytic activity was observed. The results exhibited that the main roles of extracellular proteins in activated sludges were organic molecules binding, multivalence cations as well as in the catalysis and degradation. Park et al.[54] extracted proteins present in the extracellular polymeric substances (EPS) of four full-scale activated sludge flocs using three cation-associated extraction methods. The subproteome generated from activated sludges were then fractionated by ammonium sulfate precipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that each extraction method led to unique SDS-PAGE protein profiles, which provided strong facts that the extracted proteins are exceptionally associated with specific cations in activated sludge flocs.[33]