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Protein Engineering and Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
Database searching aims to fasten the identification process and assist in accurate identification for large numbers of proteins. Different database processes such as peptide mass fingerprinting, amino acid sequence, and de novo peptide sequence are being utilized for identification of proteins. In peptide mass fingerprinting, masses of peptides from the proteolysis of target proteins are compared to the predicted masses of peptides from the theoretical digestion of proteins in a database. Amino acid sequence database searching employs identification of amino acid sequence by interpretation of the MS spectrum, which is further employed to identify unknown peptide sequence based on the molecular masses. De novo peptide sequence approach is based on identification of de novo sequence data from peptides by MS/MS and utilization of the peptide sequence to search appropriate database. This method is particularly suitable in the case that lacks well-annotated databases such as in Xenopus laevis or human. Different uninterpreted MS/MS data searching program such as Mascot, SONAR, and SEQUEST are also available for the identification of proteins (Graves and Haystead 2002).
Differential Protein Expression Following JP-8 Jet Fuel Exposure
Published in Mark L. Witten, Errol Zeiger, Glenn D. Ritchie, Jet Fuel Toxicology, 2010
Frank A. Witzmann, Mark L. Witten
More recently, owing to the high false-discovery rates associated with peptide mass fingerprinting, proteins cut from gels have been identified using tandem mass spectrometry. In this case, tryptic peptide samples are injected into a Thermo Scientific LTQ linear ion trap mass spectrometer via an auto-sampler coupled to a nanoHPLC apparatus. Peptides are ionized by nanoelectrospray, and data are collected in a “Triple-Play” (MS scan, Zoom scan, and MS/MS scan). The acquired mass spectral data are then searched against a FASTA format database (corresponding to the species used in the experiment) assembled using gene annotations publicly available from PIR (Protein Information Resource, http://pir.georgetown.edu) using the SEQUEST (v. 28 rev. 12) program in Bioworks (v. 3.3). To avoid false-positive identifications, the searched peptides and proteins are validated via PeptideProphet and ProteinProphet in the Trans-Proteomic Pipeline (TPP) (http://tools.proteomecenter.org/software.php), and only those proteins with greater than 90% confidence (containing multiple peptides with greater than 90% confidence) are considered positive identifications.
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.
Toxicoproteomic assessment of liver responses to acute pyrrolizidine alkaloid intoxication in rats
Published in Journal of Environmental Science and Health, Part C, 2018
Yan-Hong Li, William Chi-Shing Tai, Imran Khan, Cheng Lu, Yao Lu, Wing-Yan Wong, Wood-Yee Chan, Wen-Luan Wendy Hsiao, Ge Lin
Individual protein spots of interest were excised from gels, de-stained, and dehydrated before being subjected to trypsin (Promega, Madison, WI) digestion. Peptides were then extracted, vacuum-dried, and stored at −20°C prior to mass spectrometry (MS) analysis. Proteins were identified using an Autoflex III MALDI TOF/TOF MS (Bruker, Germany) equipped with a 200 Hz N2 laser (λ = 337 nm). Data were acquired in the positive ion reflector mode over a mass range of 800–4,000 Da. External calibration was performed using peptide calibration standards (Bruker Daltonics). Keratin contamination peaks, matrix ion peaks, and trypsin ion peaks were excluded from spectra. Typically 400 shots were accumulated per spectrum in MS mode and 2,000 shots in MS/MS mode. All spectra were processed using FlexAnalysis 3.0 and BioTools 3.1 software tools (Bruker, Germany). The peptide mass fingerprinting was compared with the International Protein Index database using the Mascot search program (version 2.2.04, http://www.matrixscience.com) to identify the corresponding proteins. Parameters for database searches are as follows: monoisotopic mass accuracy < 100 ppm; missed cleavages up to 1; carbamido methylation of cysteine as fixed modification; and the oxidation of methionine as variable modifications. In MS/MS mode, the fragment ion mass accuracy was set to <0.5 Da.