Chymotryptic peptide mapping MCQs With Answer

Introduction

Chymotryptic peptide mapping is an essential analytical technique in protein characterization and formulation studies for M. Pharm students. This blog provides targeted multiple-choice questions that explore chymotrypsin specificity, digestion conditions, sample preparation (denaturation, reduction, alkylation), effects of post-translational modifications, mass spectrometry readouts, and troubleshooting common problems like missed cleavages and autolysis. Understanding chymotryptic mapping helps in identifying sequence coverage, locating modifications, and designing orthogonal proteolytic strategies often complementary to trypsin. These MCQs are designed to deepen conceptual knowledge and practical skills necessary for implementation of robust peptide mapping workflows in pharmaceutical research and quality control.

Q1. What is the primary cleavage specificity of chymotrypsin during peptide mapping?

• Cleaves on the amino side of basic residues (Lys, Arg)
• Cleaves on the carboxyl side of aromatic residues (Phe, Tyr, Trp)
• Non-specific endopeptidase that cleaves randomly
• Cleaves only at glycosylation sites

Correct Answer: Cleaves on the carboxyl side of aromatic residues (Phe, Tyr, Trp)

Q2. Which statement best describes the S1 pocket preference of chymotrypsin?

• S1 pocket prefers small polar residues like Ser and Thr
• S1 pocket is hydrophobic and accommodates aromatic side chains
• S1 pocket strongly favors negatively charged residues
• S1 pocket is shaped to bind proline residues exclusively

Correct Answer: S1 pocket is hydrophobic and accommodates aromatic side chains

Q3. Which pre-digestion step most improves chymotryptic access to internal cleavage sites in a disulfide-stabilized protein?

• Incubation with EDTA to chelate metal ions
• Reduction of disulfide bonds and alkylation of cysteines
• Glycan removal by PNGase F only
• Heat denaturation without chemical reagents

Correct Answer: Reduction of disulfide bonds and alkylation of cysteines

Q4. Typical optimal pH used for chymotryptic digestion in peptide mapping workflows is:

• pH 2.0–3.0
• pH 5.0–6.0
• pH 7.5–8.5
• pH 10.5–11.5

Correct Answer: pH 7.5–8.5

Q5. Which reagent is commonly used to quench chymotrypsin digestion prior to LC-MS analysis?

• Sodium hydroxide to raise pH
• Formic acid or trifluoroacetic acid to acidify the sample
• Additional chymotrypsin to increase digestion
• EDTA to chelate divalent ions

Correct Answer: Formic acid or trifluoroacetic acid to acidify the sample

Q6. Which factor most commonly causes missed cleavages in chymotryptic peptide mapping?

• Excess protease-to-substrate ratio
• Adjacent proline residues or steric hindrance at the cleavage site
• High MS instrument resolution
• Use of sequencing-grade chymotrypsin

Correct Answer: Adjacent proline residues or steric hindrance at the cleavage site

Q7. When complementing tryptic maps, why is chymotryptic digestion often employed?

• Chymotrypsin produces identical cleavage patterns to trypsin
• Chymotrypsin provides orthogonal cleavage specificity increasing sequence coverage
• Chymotrypsin only cleaves glycosylation sites which trypsin cannot
• Chymotrypsin inactivates post-translational modifications

Correct Answer: Chymotrypsin provides orthogonal cleavage specificity increasing sequence coverage

Q8. Which of the following modifications can hinder chymotryptic cleavage and complicate mapping?

• Oxidation of methionine distant from aromatic residues
• Glycosylation near an aromatic cleavage site
• Acetylation of the protein N-terminus only
• Reduction of disulfides without alkylation

Correct Answer: Glycosylation near an aromatic cleavage site

Q9. Which enzyme treatment is used to generate active chymotrypsin from its zymogen chymotrypsinogen during preparation?

• Autoactivation at acidic pH without proteases
• Proteolytic activation by limited trypsin cleavage
• Incubation with PNGase F
• Reduction with DTT

Correct Answer: Proteolytic activation by limited trypsin cleavage

Q10. What is a common enzyme-to-substrate (E:S) ratio used for chymotryptic digestion in peptide mapping?

• 1:1 (equal weights)
• 1:10,000 (very low enzyme)
• 1:50 to 1:100 (w/w or molar depending on protocol)
• 10:1 (enzyme excess)

Correct Answer: 1:50 to 1:100 (w/w or molar depending on protocol)

Q11. Which of the following is a major drawback of chymotrypsin compared to trypsin for routine high-throughput mapping?

• Chymotrypsin produces peptides that are always too small for MS analysis
• Chymotrypsin exhibits broader specificity and often more variable cleavage, leading to complex peptide mixtures
• Chymotrypsin cannot be used with mass spectrometry
• Chymotrypsin only cleaves at acidic residues

Correct Answer: Chymotrypsin exhibits broader specificity and often more variable cleavage, leading to complex peptide mixtures

Q12. Which condition can inactivate chymotrypsin during sample preparation if not controlled?

• Maintaining pH at 8.0
• Storage or incubation in high concentrations of guanidine HCl without dilution
• Using ammonium bicarbonate buffer
• Dilution to reduce denaturant concentration prior to digestion

Correct Answer: Storage or incubation in high concentrations of guanidine HCl without dilution

Q13. How does alkylation (e.g., iodoacetamide) after reduction improve chymotryptic peptide mapping?

• It increases protease activity directly
• It prevents reformation of disulfide bonds, maintaining peptide accessibility for digestion
• It removes glycosyl groups that block cleavage
• It fragments the protein chemically to mimic enzymatic cleavage

Correct Answer: It prevents reformation of disulfide bonds, maintaining peptide accessibility for digestion

Q14. Autolytic peptides from chymotrypsin can interfere with LC-MS maps. Which approach reduces autolysis-derived background?

• Increase digestion time to overnight
• Use sequencing-grade chymotrypsin and optimize enzyme amount and digestion time
• Avoid reduction and alkylation steps
• Perform digestion at very high temperature (95°C)

Correct Answer: Use sequencing-grade chymotrypsin and optimize enzyme amount and digestion time

Q15. In LC-MS peptide mapping, which peptide mass range is typically most suitable for confident identification following chymotryptic digestion?

• 100–300 Da
• 500–3000 Da
• 10,000–50,000 Da
• >100,000 Da

Correct Answer: 500–3000 Da

Q16. Which amino acid at the P1′ position commonly reduces chymotryptic cleavage efficiency?

• Alanine
• Glycine
• Proline
• Leucine

Correct Answer: Proline

Q17. Why might one perform limited chymotryptic digestion (short incubations) in mapping workflows?

• To intentionally generate only very small dipeptides
• To produce overlapping peptides that aid in mapping PTMs and sequence confirmation
• To completely denature the protein irreversibly
• To hydrolyze only glycosidic bonds

Correct Answer: To produce overlapping peptides that aid in mapping PTMs and sequence confirmation

Q18. Which mass spectrometric evidence supports identification of a chymotryptic peptide that carries an oxidized methionine?

• No mass shift compared to unmodified peptide
• A mass increase of +16 Da on the peptide ion corresponding to oxygen addition
• A mass decrease of -18 Da due to loss of water
• Complete absence of the peptide signal always

Correct Answer: A mass increase of +16 Da on the peptide ion corresponding to oxygen addition

Q19. When using urea for denaturation prior to chymotrypsin digestion, what precaution is important?

• Keep urea concentration >8 M during digestion to enhance cleavage
• Dilute urea to a concentration compatible with enzyme activity (often <2 M) before adding chymotrypsin
• Avoid reduction and alkylation if urea is present
• Always boil the sample with urea at 100°C during digestion

Correct Answer: Dilute urea to a concentration compatible with enzyme activity (often <2 M) before adding chymotrypsin

Q20. Which analytical outcome specifically indicates improved sequence coverage after performing chymotryptic mapping in addition to tryptic mapping?

• Decreased number of identified peptides overall
• Identification of peptides spanning regions that were not observed in tryptic maps, increasing overall sequence coverage percentage
• Loss of detection for all glycopeptides
• Exclusive identification of single amino acids

Correct Answer: Identification of peptides spanning regions that were not observed in tryptic maps, increasing overall sequence coverage percentage

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