Tryptic peptide mapping MCQs With Answer

Tryptic peptide mapping MCQs With Answer is a concise, practice-focused quiz designed for M.Pharm students studying Proteins and Protein Formulations. This set emphasizes practical and theoretical aspects of tryptic digestion, sample preparation, analytical techniques (HPLC/LC‑MS/MS), and interpretation of peptide maps for biopharmaceutical characterization. Questions address enzymatic specificity, buffer systems, reduction/alkylation strategies, artifacts, digestion kinetics, and analytical troubleshooting—topics critical for identity, sequence coverage, and PTM monitoring in protein therapeutics. Each question is crafted to reinforce lab-relevant decision making and to deepen understanding of how experimental variables influence peptide mapping outcomes and data quality.

Q1. Which peptide bond specificity best describes trypsin cleavage?

• Cleave C‑terminal to lysine and arginine residues, except when followed by proline
• Cleave N‑terminal to aromatic residues such as phenylalanine and tyrosine
• Cleave only after cysteine residues
• Cleave randomly at internal peptide bonds without sequence specificity

Correct Answer: Cleave C‑terminal to lysine and arginine residues, except when followed by proline

Q2. What is the commonly used pH range for optimal trypsin activity during peptide mapping?

• pH 3.0–4.0 (acidic)
• pH 5.5–6.5 (slightly acidic)
• pH 7.5–8.5 (commonly ~8.0)
• pH 10.0–11.0 (strongly basic)

Correct Answer: pH 7.5–8.5 (commonly ~8.0)

Q3. Why are reduction and alkylation steps performed prior to tryptic digestion in peptide mapping workflows?

• To hydrolyze peptide bonds selectively at aromatic residues
• To reduce disulfide bonds and alkylate cysteines to prevent reformation, improving digestion and peptide recovery
• To increase protein glycosylation for better MS detection
• To remove N‑terminal acetylation from peptides

Correct Answer: To reduce disulfide bonds and alkylate cysteines to prevent reformation, improving digestion and peptide recovery

Q4. Which alkylating reagent is most commonly used to modify free cysteines after reduction in peptide mapping protocols?

• Iodoacetamide (IAA)
• Sodium borohydride
• Hydrazine
• Formaldehyde

Correct Answer: Iodoacetamide (IAA)

Q5. A typical enzyme:substrate (E:S) ratio for trypsin digestion by weight in peptide mapping is:

• 1:1 (equal weights)
• 1:10,000 (very low enzyme)
• 1:20 to 1:100 (w/w)
• 10:1 (excess enzyme)

Correct Answer: 1:20 to 1:100 (w/w)

Q6. What is a commonly used incubation condition for trypsin digestion to achieve good sequence coverage?

• 4°C overnight to preserve enzyme stability
• Room temperature for 1 hour only
• 37°C for ~4–16 hours (overnight is common)
• 95°C for 10 minutes to accelerate cleavage

Correct Answer: 37°C for ~4–16 hours (overnight is common)

Q7. Which structural feature commonly causes missed cleavages by trypsin?

• Presence of a proline residue immediately following lysine or arginine (P1′ position)
• An alanine residue at P1′ position
• High content of glycine throughout the protein
• Low molecular weight peptides only

Correct Answer: Presence of a proline residue immediately following lysine or arginine (P1′ position)

Q8. Which buffer is preferred for trypsin digestion prior to LC‑MS because it is volatile and mass‑spec compatible?

• Phosphate-buffered saline (PBS)
• Tris-HCl (non‑volatile)
• Ammonium bicarbonate (volatile)
• Sodium acetate

Correct Answer: Ammonium bicarbonate (volatile)

Q9. How is enzymatic digestion typically quenched before LC‑MS analysis?

• By heating to 100°C for 1 minute
• By diluting in water to reduce enzyme concentration
• By acidifying with formic acid or TFA to pH <3
• By adding more trypsin inhibitor buffer at pH 8

Correct Answer: By acidifying with formic acid or TFA to pH <3

Q10. Which analytical technique provides sequence confirmation and PTM localization from tryptic peptide maps?

• UV spectrophotometry of intact protein
• Size‑exclusion chromatography (SEC)
• LC‑MS/MS (tandem mass spectrometry)
• SDS‑PAGE gel staining

Correct Answer: LC‑MS/MS (tandem mass spectrometry)

Q11. What is the primary purpose of tryptic peptide mapping in biopharmaceutical analysis?

• To determine protein tertiary structure by circular dichroism
• To confirm primary amino acid sequence, detect PTMs and assess comparability/stability
• To measure protein solubility in different buffers
• To quantify intact protein molecular weight by gel electrophoresis only

Correct Answer: To confirm primary amino acid sequence, detect PTMs and assess comparability/stability

Q12. In peptide mapping, what does “sequence coverage” refer to?

• The proportion of identified peptides that are hydrophobic
• The percentage of the protein’s amino acid sequence represented by identified peptides
• The number of peptides detected per minute during chromatography
• Total mass of peptides recovered after digestion

Correct Answer: The percentage of the protein’s amino acid sequence represented by identified peptides

Q13. How does glycosylation commonly affect tryptic peptide mapping?

• It uniformly increases cleavage efficiency at all sites
• It has no effect on peptide ionization or chromatographic behavior
• It can hinder cleavage near glycosylation sites and create heterogeneous masses, complicating analysis
• It converts lysines to arginines, altering cleavage specificity

Correct Answer: It can hinder cleavage near glycosylation sites and create heterogeneous masses, complicating analysis

Q14. What artifact can arise from using high concentrations of urea during sample preparation, and how does it form?

• Carbamylation of amines due to isocyanate from urea decomposition
• Deamidation due to urea acting as an acid catalyst
• Oxidation of methionine by urea radicals
• Glycation of lysines by urea sugars

Correct Answer: Carbamylation of amines due to isocyanate from urea decomposition

Q15. Why is desalting (e.g., C18 cleanup) recommended before LC‑MS analysis of tryptic digests?

• To increase sample volume for injection
• To remove salts and detergents that suppress electrospray ionization and interfere with LC separation
• To chemically modify peptides for better UV detection
• To neutralize basic residues on peptides

Correct Answer: To remove salts and detergents that suppress electrospray ionization and interfere with LC separation

Q16. How are missed cleavages typically identified in LC‑MS peptide maps?

• As shorter peptides than predicted by trypsin specificity
• By the absence of any signal in MS spectra
• As larger peptides containing internal lysine or arginine that correspond to contiguous tryptic sites
• By unexpected glycan masses only

Correct Answer: As larger peptides containing internal lysine or arginine that correspond to contiguous tryptic sites

Q17. When might Lys‑C be chosen as an alternative to trypsin in peptide mapping?

• When cleavage after arginine is specifically required
• For proteins with extensive Arg sites or when higher denaturant concentrations are used, since Lys‑C cleaves only after lysine and tolerates denaturants
• When one wants cleavage exclusively at aromatic residues
• When preventing cleavage at lysine is desired

Correct Answer: For proteins with extensive Arg sites or when higher denaturant concentrations are used, since Lys‑C cleaves only after lysine and tolerates denaturants

Q18. Which of the following is a specific natural inhibitor of trypsin that is sometimes used to stop proteolysis?

• Aprotinin
• PMSF (phenylmethylsulfonyl fluoride) only
• EDTA (a metal chelator)
• Urea

Correct Answer: Aprotinin

Q19. Alkylation with iodoacetamide results in what mass modification on cysteine residues that is commonly monitored in MS?

• Oxidation of +16 Da
• Carbamidomethylation of +57.021 Da
• Acetylation of +42 Da
• Phosphorylation of +80 Da

Correct Answer: Carbamidomethylation of +57.021 Da

Q20. Why are tracked changes in the abundance of specific missed‑cleavage peptides useful in stability or comparability studies of biologics?

• They only indicate instrument variability and are not biologically meaningful
• They can reflect conformational changes or PTMs that affect local protease accessibility, serving as sensitive indicators of structural changes
• They measure overall protein concentration directly
• They indicate buffer pH only and are irrelevant to structure

Correct Answer: They can reflect conformational changes or PTMs that affect local protease accessibility, serving as sensitive indicators of structural changes

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