This set of Bioinformatics Multiple Choice Questions & Answers (MCQs) focuses on “Technology of Protein Expression Analysis”.
1. The classic protein separation methods involve two-dimensional gel electrophoresis followed by gel image analysis.
Explanation: Further characterization involves determination of amino acid composition, peptide mass fingerprints, and sequences using mass spectrometry (MS). Finally, database searching is needed for protein identification.
2. Which of the following is incorrect regarding 2D-Page?
a) It stands for Two-dimensional polyacrylamide gel electrophoresis
b) It separates proteins by charge only
c) The gel is run in one direction in a pH gradient under a non-denaturing condition
d) It works to separate proteins by isoelectric points (pI)
Explanation: it is a high-resolution technique that separates proteins by charge and mass. It works to separate proteins by isoelectric points (pI) and then in an orthogonal dimension under a denaturing condition to separate proteins by molecular weights (MW). This is followed by staining, usually silver staining, which is very sensitive, to reveal the position of all proteins. The result is a two-dimensional gel map; each spot on the map corresponds to a single protein being expressed.
3. Which of the following is incorrect regarding 2D-Page?
a) Not all proteins can be separated by this method or stained properly
b) The stained gel can be scanned and digitized for image analysis
c) Membrane proteins are largely hydrophilic and readily solubilized
d) One of the challenges of this technique is the separation of membrane proteins
Explanation: membrane proteins are largely hydrophobic and not readily solublized. They tend to aggregate in the aqueous medium of a two-dimensional gel. To overcome this problem, membrane proteins can be fractionated using specialized protocols and then electrophoresed using optimized buffers containing zwitterionic detergents. Subfractionation can be carried out to separate nuclear, cytosol, cytoskeletal, and other subcellular fractions to boost the concentrations of rare proteins and to reveal subcellular localizations of the proteins.
4. Comparing two-dimensional gel images from various experiments can sometimes pose a challenge because the gels, unlike DNA microarrays, may shrink or warp.
Explanation: This requires the software programs to be able to stretch or maneuver one of the gels relative to the other to find a common geometry. When the reference spots are aligned properly, the rest of the spots can be subsequently compared automatically.
5. Which of the following is incorrect regarding Mass Spectrometry Protein Identification?
a) The proteolysis doesn’t generate a pattern according to molecular weight
b) Proteins can be identified and characterized using MS
c) The proteins from a two dimensional gel system are first digested in situ with a protease
d) Protein spots of interest are excised from the two-dimensional gel
Explanation: The proteolysis generates a unique pattern of peptide fragments of various MWs, which is termed a peptide fingerprint. The fragments can be analyzed with MS, a high-resolution technique for determining molecular masses. Currently, electro-spray ionization MS and matrix-assisted laser desorption ionization (MALDI) MS are commonly used.
6. Electrospray ionization MS and matrix-assisted laser desorption ionization (MALDI) MS only differ in the ionization procedure used.
Explanation: In MALDI-MS, for example, the peptides are charged with positive ions and forced through an analyzing tube with a magnetic field. Peptides are analyzed in the gas phase. Because smaller peptides are deflected more than larger ones in a magnetic field, the peptide fragments can be separated according to molecular mass and charges. A detector generates a spectrum that displays ion intensity as a function of the mass-to-charge ratio.
7. Which of the following is incorrect regarding the Protein Identification through Database Searching?
a) MS characterization of proteins is highly dependent on bioinformatic analysis
b) Bioinformatics programs can be used to search for the identity of a protein in a database of theoretically digested proteins
c) Even in reality, the protease digestion is always perfect in MS
d) The purpose of the database search is to find exact or nearly exact matches
Explanation: in reality, protease digestion is rarely perfect, often generating partially digested products as a result of missed cuts at expected cutting sites. Peptides resulting from MALDI-MS are also charged, which increases their mass slightly.
8. ExPASY is a comprehensive proteomics web server with a suite of programs for searching peptide information from the SWISS-PROT and TrEMBL databases.
Explanation: There are twelve database search tools in this server dedicated to protein identification based on MS data. For example, the AACompIdent program identifies proteins based on pI, MW, and amino acid composition and compares these values with theoretical compositions of all proteins in SWISS-PROT/TrEMBL.
9. Which of the following is incorrect regarding Mascot and ProFound?
a) ProFound is a web server with a set of interconnected programs
b) ProFound searches a protein sequence database using MS fingerprinting information
c) Bayesian algorithm is not involved in ProFound
d) Mascot is a web server that identifies proteins based on peptide mass fingerprints, sequence entries, or raw MS/MS data from one or more peptides
Explanation: In ProFound, A Bayesian algorithm is used. It ranks the database matches according to the probability of database sequences producing the peptide mass fingerprints.
10. Which of the following is incorrect regarding Differential In-Gel Electrophoresis?
a) Proteins are mixed together before electrophoresis on a two-dimensional gel
b) Differentially expressed proteins in both conditions can’t be visualized in the same gel
c) In this, Differences in protein expression patterns can be detected in a similar way as in fluorescent-labeled DNA microarrays
d) Proteins from experimental and control samples are labeled with differently colored fluorescent dyes
Explanation: Differentially expressed proteins in both conditions can be co-separated and visualized in the same gel. Compared to regular 2D-PAGE, the process reduces the noise and improves the reproducibility and sensitivity of detection. In principle, it resembles the two-color DNA microarray analysis. The drawbacks of this approach are that different proteins take up fluorescent tags to different extents and that some proteins labeled with the fluorophores may become less soluble and precipitate before electrophoresis.
Sanfoundry Global Education & Learning Series – Bioinformatics.
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