Dengue Fever: A Viral Hemorrhagic Fever of Global Concern
Jagriti Narang, Manika Khanuja in Small Bite, Big Threat, 2020
Genome sequencing of dengue virus: The entire genetic information of all serotypes of dengue (Angel et al., 2016; Anoop et al., 2012; Chao et al., 2005; Dayaraj et al., 2011; Ong et al., 2008; Patil et al., 2011; Schreiber et al., 2009; Sharma et al., 2011; Shin et al., 2013) and partial sequences (Angel et al., 2016; Domingo et al., 2006; Kukreti et al., 2008, 2009) have been investigated. Table 4.6 depicts the results of protein sequencing. The genetic material of DENV is RNA and, thus, mutates at a fast rate (Drake, 1993; Holmes and Burch, 2000; Bennett et al., 2003). Chin-Inmanu et al. (2012) have used pyro-sequencing strategy for sequencing the viral genome. The Broad Institute Massachusetts, USA, and many pioneer institutes have contributed to huge data of DENV genome (Ong, 2010).
Chemical Methods of Amino Acid Sequence Analysis from Carboxy Terminal End
Ajit S. Bhown in Protein/Peptide Sequence Analysis: Current Methodologies, 1988
One of the eaR1iest chemical methods for the sequential degradation of peptides from the C terminus was proposed by Bergmann and Zervas.3 They converted N-benzoyl peptide ester into N-benzoyl peptide hydrazide by treatment with hydrazine. The hydrazide was converted to the azide by reacting with nitrous acid, as shown in Figure 1. On treating the azide with benzyl alcohol, it was converted into the benzyl urethane derivative. Catalytic reduction of the N-benzoyl peptide benzyl urethane derivative gave two products, i.e., N-benzoyl peptide_1 amide and the original C-terminal amino acid as its aldehyde. The method did not gain popularity because of the extreme complexity of the chemical reactions involved. Several years later, Khorana4 proposed another very complicated set of reactions for the sequential degradation of peptides. The method involved reacting an acyl peptide such as N-benzoyl peptide with a disubstituted carbodi-imide to yield the acyl-peptidyl urea. This set of reactions is set out in Figure 2. Treatment of the acyl-peptidyl urea with ethanolic-sodium hydroxide yielded the shortened peptide and a substituted carbamyl amino acid amide. The amino acid derivative could be identified by hydrolyzing it to the parent amino acid with acid or alkali. Neither of the two methods described so far gained ground in the field of protein sequencing, because of the extreme complexity of the reactions involved and the incomplete removal of the C-terminal amino acid after each cycle.
The Use of Sperm Proteomics in the Assisted Reproduction Laboratory
Nicolás Garrido, Rocio Rivera in A Practical Guide to Sperm Analysis, 2017
The study of sperm proteins started more than a century ago with the isolation and identification by Friedrich Miescher in 1874 of a proteinaceous basic component from the sperm cell that he called “protamine” and that he found was coupled to what he called “nuclein” or what we know as DNA.13 However, it was not until about 100 years later that the protein sequencing, separation, and detection methods were developed allowing the generalized study of the proteins (Figure 18.1).14–16 Nevertheless, with these methods the proteins still had to be studied one at a time. The possibility to study the entire or a substantial proportion of the sperm proteome started much more recently, around 1995, with the application of mass spectrometry to the study of proteins (Figure 18.1).
De novo sequencing of proteins by mass spectrometry
Published in Expert Review of Proteomics, 2020
Rui Vitorino, Sofia Guedes, Fabio Trindade, Inês Correia, Gabriela Moura, Paulo Carvalho, Manuel A. S. Santos, Francisco Amado
The advent of MS for protein identification, and its combination with de novo sequencing has revolutionized modern proteomics with the development of the nascent field of proteogenomics. The ability to identify novel peptides, their sequences, mutations, and modifications using these advanced techniques has broadened the understanding of molecular biology, particularly of proteins. Unraveling the genomic features of a protein is crucial for their characterization and understanding their functional role. There are various advancements in de novo sequencing of proteins, and new software are being developed continuously for robust and accurate identification. The Human Genome Project has paved the way for in-silico studies that will save time and optimize the use of resources. The limitations of such techniques are being studied to improve the usefulness of this approach in protein sequencing.
Recombinant human C1 esterase inhibitor (Conestat alfa) for prophylaxis to prevent attacks in adult and adolescent patients with hereditary angioedema
Published in Expert Review of Clinical Immunology, 2018
Anna Valerieva, Sonia Caccia, Marco Cicardi
Conestat alpha is a rhC1-INH (Ruconest®, Pharming Technologies B.V., Leiden, The Netherlands) obtained through a purification process of transgenic New Zealand white rabbits’ milk (Oryctolagus cuniculus). The promoter used to drive expression of the hC1-INH transgene is the bovine alpha-S1-casein promoter, which is specific for the secretion of caseins in the milk. The protein content of rabbit milk is approximately 14% of which about 65% consists of various caseins aggregated in micelles. The remaining proteins in rabbit milk are whey proteins including transferrin, whey acidic protein, immunoglobulins, albumin, and lactalbumin. After collection, rabbit milk undergoes series of standard centrifugation, filtration, and chromatography steps that give rhC1-INH, 99% purity as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis [43]. The 1% impurities are multimers and N-terminal cleaved C1-INH species. Host-related impurities were analyzed (ELISA) and measured to be approximately 10 ppm in the commercial batches and defined to consist of minimal traces of rabbit protein [43]. The activity of purified rhC1-INH is 6.1 U per mg of protein, the same as pdC1-INH [43]. Protein sequencing analysis of recombinant and plasma proteins reveals identical polypeptide N- and C-terminals for the two molecules. Nevertheless, the two proteins differ in molecular mass and this is explained by differences in glycosylation (21% and 26–28% carbohydrate content respectively) [43,44].
Proteomics of Pseudomonas aeruginosa: the increasing role of post-translational modifications
Published in Expert Review of Proteomics, 2018
Charlotte Gaviard, Thierry Jouenne, Julie Hardouin
An interesting alternative to bottom–up is top–down proteomics (Figure 1), recently reviewed [62]. Intact proteins are directly analyzed, without enzymatic digestion [63]. Fractionation steps are first included in the workflow, to decrease protein mixture complexity and to improve top–down analyses of whole proteins. Protein fractionation can be performed by LC, IEF, or capillary zone electrophoresis (CZE) but one-dimensional separation may be not enough efficient. Li et al. optimized capillary electrophoresis and identify 30 intact proteins of P. aeruginosa strain PAO1 by top–down proteomics [64]. Two-dimensional [65] or even four-dimensional [66] prefractionation has been described to enhance protein separation. Then, the use of high-resolution and high mass accuracy mass spectrometers is crucial to distinguish signals of the different proteoforms that can just exhibit mass differences below 0.01 Da. The precise mass of the intact proteins is determined and protein sequencing is achieved by MS/MS using ECD or ETD on FT-ICR or orbitrap mass spectrometers, respectively.
Related Knowledge Centers
- Hydrolysis
- Peptide
- Protease
- Protein
- Protein Primary Structure
- Post-Translational Modification
- Translation
- Gene
- N-Terminus
- Hydrochloric Acid