De novo protein characterization MCQs With Answer

De novo protein characterization MCQs With Answer

Introduction

This quiz set focuses on de novo protein characterization—an essential area for M.Pharm students involved in biologics, biosimilars and peptide drug development. It covers analytical strategies used when sequence information is unknown or when studying novel/engineered proteins: mass spectrometry-based de novo sequencing, Edman degradation, fragmentation ion interpretation, PTM mapping, and orthogonal biophysical methods for structure and stability. Questions also integrate formulation-relevant characterization: aggregation assessment, thermal stability, and sample preparation challenges. The goal is to deepen understanding of techniques, data interpretation, limitations and practical considerations needed for rigorous characterization of new proteins in a pharmaceutical context.

Q1. Which technique is most suitable for determining the amino acid sequence of a novel protein when no database entry is available?

• Edman degradation
• Tandem mass spectrometry de novo sequencing
• X-ray crystallography
• Differential scanning calorimetry (DSC)

Correct Answer: Tandem mass spectrometry de novo sequencing

Q2. During MS/MS de novo peptide sequencing, which ion series is typically used to identify residues from the N-terminus?

• y-ions
• b-ions
• a-ions
• z-ions

Correct Answer: b-ions

Q3. A common limitation in de novo MS-based sequencing is the inability to distinguish isobaric residues. Which pair is isobaric and therefore difficult to differentiate by mass alone?

• Serine and Threonine
• Leucine and Isoleucine
• Lysine and Arginine
• Aspartic acid and Asparagine

Correct Answer: Leucine and Isoleucine

Q4. Which fragmentation method is preferred for preserving labile post-translational modifications (PTMs) such as phosphorylation during MS/MS?

• Collision-induced dissociation (CID)
• Higher-energy collisional dissociation (HCD)
• Electron transfer dissociation (ETD)
• In-source decay

Correct Answer: Electron transfer dissociation (ETD)

Q5. In bottom-up de novo sequencing workflows, what is the primary purpose of reduction and alkylation of cysteines before proteolysis?

• Enhance enzymatic activity of proteases
• Prevent reformation of disulfide bonds and simplify peptide mapping
• Label peptides for quantitation
• Increase hydrophobicity for reversed-phase separation

Correct Answer: Prevent reformation of disulfide bonds and simplify peptide mapping

Q6. Which protease specificity is most commonly used for peptide generation in de novo sequencing workflows because it produces peptides of convenient length?

• Chymotrypsin (after aromatic residues)
• Trypsin (after Lys/Arg)
• Glu-C (after Glu)
• Pepsin (acidic conditions)

Correct Answer: Trypsin (after Lys/Arg)

Q7. When performing intact mass analysis for a novel protein (top-down MS), which information can be obtained directly that is often missed in bottom-up approaches?

• Primary sequence of every peptide fragment
• Overall intact molecular weight and combination of PTMs on the same proteoform
• Secondary structure content
• Hydrodynamic radius

Correct Answer: Overall intact molecular weight and combination of PTMs on the same proteoform

Q8. In de novo peptide sequencing using MS/MS, what does a mass shift of approximately +79.97 Da on a residue typically indicate?

• Oxidation
• Methylation
• Phosphorylation
• Acetylation

Correct Answer: Phosphorylation

Q9. Which orthogonal technique provides information about secondary structure composition (alpha-helix, beta-sheet) useful for characterizing novel proteins and confirming folded state?

• Dynamic light scattering (DLS)
• Circular dichroism (CD) spectroscopy
• Size-exclusion chromatography (SEC)
• Mass spectrometry

Correct Answer: Circular dichroism (CD) spectroscopy

Q10. During peptide de novo sequencing, what is the major practical consequence of missed protease cleavage sites?

• Improved sequence coverage due to longer peptides
• Generation of unexpected peptide masses complicating spectrum interpretation
• Complete protection of PTMs from fragmentation
• Increased ionization efficiency uniformly across peptides

Correct Answer: Generation of unexpected peptide masses complicating spectrum interpretation

Q11. Which technique is most appropriate for detecting and quantifying soluble oligomeric aggregates of a biologic during formulation studies?

• Reversed-phase HPLC
• Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS)
• Edman degradation
• Native gel electrophoresis without standards

Correct Answer: Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS)

Q12. Which MS-based approach allows mapping of disulfide bonds in a de novo characterized protein?

• Intact mass measurement without reduction only
• Non-reducing SDS-PAGE
• Targeted peptide mapping comparing reduced versus non-reduced proteolytic digests by LC-MS/MS
• Circular dichroism spectroscopy

Correct Answer: Targeted peptide mapping comparing reduced versus non-reduced proteolytic digests by LC-MS/MS

Q13. What is a key advantage of de novo sequencing algorithms (e.g., PEAKS, Novor) over database-search methods?

• They are faster for very large proteomes
• They can identify peptide sequences without any prior sequence database
• They require less high-resolution mass spectrometers
• They always yield 100% accurate full-length protein sequences

Correct Answer: They can identify peptide sequences without any prior sequence database

Q14. In stress testing for formulation development of a novel protein, which analytical method provides the most direct measurement of thermal stability (unfolding temperature)?

• Dynamic light scattering (DLS)
• Differential scanning calorimetry (DSC)
• Mass spectrometry intact mass
• Isoelectric focusing

Correct Answer: Differential scanning calorimetry (DSC)

Q15. Which MS fragmentation behavior would you expect when using collision-induced dissociation (CID) on a typical peptide?

• Cleavage predominantly along the peptide backbone to produce b- and y-ions
• Exclusive preservation of intact precursor ions
• Predominant cleavage of glycosidic bonds only
• Random fragmentation giving no interpretable series

Correct Answer: Cleavage predominantly along the peptide backbone to produce b- and y-ions

Q16. For de novo glycopeptide characterization, what is one major analytical challenge compared to non-glycosylated peptides?

• Glycopeptides ionize much better and dominate spectra
• Glycan heterogeneity causes multiple isobaric/near-isobaric species and complex fragmentation patterns
• Glycopeptides cannot be separated by liquid chromatography
• Glycosylation prevents enzymatic digestion completely

Correct Answer: Glycan heterogeneity causes multiple isobaric/near-isobaric species and complex fragmentation patterns

Q17. Which method is particularly useful for assessing hydrodynamic radius and polydispersity of protein samples during formulation screening?

• Capillary isoelectric focusing
• Dynamic light scattering (DLS)
• Edman degradation
• MALDI-TOF peptide mass fingerprinting

Correct Answer: Dynamic light scattering (DLS)

Q18. When de novo sequencing an unknown protein by bottom-up MS, why is high mass accuracy (e.g., sub-ppm) important?

• It speeds up liquid chromatography
• It reduces chemical noise in CD spectra
• It narrows candidate residue combinations and improves confidence in sequence assignments
• It eliminates the need for proteolysis

Correct Answer: It narrows candidate residue combinations and improves confidence in sequence assignments

Q19. If a de novo sequencing workflow returns a peptide containing an unexpected mass increase of +15.99 Da, which modification is most likely?

• Methylation
• Oxidation (commonly of methionine)
• Deamidation
• Phosphorylation

Correct Answer: Oxidation (commonly of methionine)

Q20. In characterizing a novel therapeutic protein, which integrated strategy best increases confidence in assigned sequence and structural features?

• Rely solely on a single high-resolution MS/MS experiment
• Combine complementary methods: bottom-up and top-down MS, orthogonal biophysical techniques (CD, DSC, SEC-MALS) and targeted enzymatic/chemical assays
• Use only Edman degradation for full-length determination
• Assess formulation behavior without sequence confirmation

Correct Answer: Combine complementary methods: bottom-up and top-down MS, orthogonal biophysical techniques (CD, DSC, SEC-MALS) and targeted enzymatic/chemical assays

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