Protein identification strategies MCQs With Answer

Protein identification strategies MCQs With Answer

This quiz collection is designed for M.Pharm students to reinforce core concepts and practical approaches used in protein identification. It covers analytical techniques such as mass spectrometry (MALDI-TOF, ESI, tandem MS/MS), Edman degradation, 2D-PAGE, Western blotting, top-down and bottom-up proteomics, peptide mass fingerprinting, and strategies for detecting post-translational modifications and glycosylation. Questions emphasize methodological principles, sample preparation, data interpretation, and common limitations encountered in pharmaceutical protein analysis. Use these MCQs to test theoretical understanding and to prepare for applied tasks in formulation development, quality control, and research where precise protein characterization is essential.

Q1. Which technique is most suitable for rapid determination of the molecular mass of an intact protein?

• SDS-PAGE with Coomassie staining
• MALDI-TOF mass spectrometry
• Edman degradation sequencing
• Circular dichroism spectroscopy

Correct Answer: MALDI-TOF mass spectrometry

Q2. What is the fundamental principle behind Edman degradation for protein sequencing?

• Hydrolysis of proteins into amino acids followed by chromatography
• Sequential N-terminal removal and identification using phenylisothiocyanate (PITC)
• Mass determination of intact proteins by ionization
• Enzymatic cleavage at specific internal residues and MS analysis

Correct Answer: Sequential N-terminal removal and identification using phenylisothiocyanate (PITC)

Q3. How does bottom-up proteomics differ from top-down proteomics?

• Bottom-up analyzes intact proteins; top-down analyzes digested peptides
• Bottom-up uses antibody detection; top-down uses chromatography only
• Bottom-up digests proteins into peptides before MS; top-down analyzes intact proteoforms
• Bottom-up detects post-translational modifications directly on intact proteins

Correct Answer: Bottom-up digests proteins into peptides before MS; top-down analyzes intact proteoforms

Q4. Peptide mass fingerprinting (PMF) primarily relies on which combination to identify proteins?

• Edman sequencing and antibody arrays
• MALDI-TOF peptide masses and database matching
• SDS-PAGE band staining intensity and migration distance
• NMR spectral fingerprints compared to standards

Correct Answer: MALDI-TOF peptide masses and database matching

Q5. Which fragmentation pattern is commonly observed in collision-induced dissociation (CID) MS/MS and useful for peptide sequencing?

• a and c ions predominating
• b and y ions predominating
• x and z ions predominating
• Intact molecular ion with no fragmentation

Correct Answer: b and y ions predominating

Q6. For sensitive identification of phosphorylation sites on peptides, which strategy is most appropriate?

• Direct Edman degradation of whole protein
• Enrichment of phosphopeptides followed by LC-MS/MS
• MALDI imaging of tissue without enrichment
• Gel filtration chromatography alone

Correct Answer: Enrichment of phosphopeptides followed by LC-MS/MS

Q7. What does a Mascot score represent in proteomics database searching?

• The absolute molecular weight of a peptide
• A statistical measure of the likelihood that a peptide-spectrum match is correct
• The chromatographic retention time of a peptide
• The concentration of protein in a sample

Correct Answer: A statistical measure of the likelihood that a peptide-spectrum match is correct

Q8. When is de novo peptide sequencing from MS/MS spectra preferred?

• When high-quality genomic databases are available
• When antibodies for the protein are readily available
• When the protein sequence is not present in any database
• When only intact mass but no fragment ions are observed

Correct Answer: When the protein sequence is not present in any database

Q9. What is the specificity of trypsin used in proteolytic digestion for MS-based identification?

• Cleaves at the N-terminal side of methionine residues
• Cleaves at the C-terminal side of lysine and arginine residues
• Randomly cleaves peptide bonds regardless of residue
• Cleaves exclusively at aromatic residues

Correct Answer: Cleaves at the C-terminal side of lysine and arginine residues

Q10. Why are reducing agents and alkylation reagents used during sample preparation for MS?

• To fluorescently label peptides for detection
• To cleave peptide bonds at methionine residues
• To reduce disulfide bonds and prevent reformation by alkylation
• To enhance protein tertiary structure for better ionization

Correct Answer: To reduce disulfide bonds and prevent reformation by alkylation

Q11. What is a principal advantage of top-down proteomics over bottom-up approaches?

• Requires no fragmentation and thus no MS instrument
• Provides information on intact proteoforms including combinations of PTMs and sequence variants
• Is less demanding in sample purity and complexity
• Always provides higher throughput than bottom-up

Correct Answer: Provides information on intact proteoforms including combinations of PTMs and sequence variants

Q12. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separates proteins based on which two properties?

• Molecular shape and hydrophobicity
• Isoelectric point (pI) and molecular weight
• Charge only in both dimensions
• Glycosylation status and phosphorylation level

Correct Answer: Isoelectric point (pI) and molecular weight

Q13. Western blotting identifies a protein primarily by which mechanism?

• Mass spectrometric mass fingerprinting
• Antibody recognition of specific epitopes
• Direct N-terminal sequencing of the protein in-gel
• Fluorescent circular dichroism signal

Correct Answer: Antibody recognition of specific epitopes

Q14. What best describes Selected Reaction Monitoring (SRM)/Multiple Reaction Monitoring (MRM) in targeted proteomics?

• Untargeted discovery of all peptides in a sample
• Monitoring predefined precursor→product ion transitions using a triple-quadrupole MS for quantitation
• Electrophoretic separation of proteins before gel staining
• A technique for de novo sequencing without MS

Correct Answer: Monitoring predefined precursor→product ion transitions using a triple-quadrupole MS for quantitation

Q15. Which enzyme is commonly used to release N-linked glycans from glycoproteins for analysis?

• Trypsin
• PNGase F
• Pepsin
• Endoproteinase Lys-C

Correct Answer: PNGase F

Q16. Isoelectric focusing separates proteins primarily by which property?

• Hydrophobicity
• Size exclusion based on molecular radius
• Net charge as a function of pH (isoelectric point)
• Affinity to lectins

Correct Answer: Net charge as a function of pH (isoelectric point)

Q17. A major limitation of Edman degradation is:

• It can sequence peptides regardless of post-translational modifications
• It cannot sequence proteins with blocked N-termini or very large proteins efficiently
• It provides high-throughput identification of complex mixtures
• It directly identifies phosphorylation sites without enrichment

Correct Answer: It cannot sequence proteins with blocked N-termini or very large proteins efficiently

Q18. What is an advantage of capillary electrophoresis (CE) in protein/peptide analysis?

• It requires large sample volumes for robust detection
• High-resolution separation with low sample consumption and fast analysis time
• Inability to be coupled to mass spectrometry
• Exclusive separation by hydrophobic interactions

Correct Answer: High-resolution separation with low sample consumption and fast analysis time

Q19. Stable isotope labeling methods like SILAC or iTRAQ are used in proteomics primarily for:

• Improving ionization efficiency of peptides
• Absolute structural determination of proteins
• Relative or absolute quantitative comparison of protein abundance across samples
• Selective enrichment of glycopeptides

Correct Answer: Relative or absolute quantitative comparison of protein abundance across samples

Q20. Circular dichroism (CD) spectroscopy is most useful in protein analysis for assessing:

• The exact amino acid sequence of a protein
• Secondary structure content and folding changes
• Precise molecular weight of intact proteins
• Peptide fragment ion masses for sequencing

Correct Answer: Secondary structure content and folding changes

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