Edman degradation MCQs With Answer

Introduction: Edman degradation MCQs With Answer is a focused practice resource designed for M.Pharm students studying Proteins and Protein Formulations. This blog concentrates on the Edman degradation method—its chemical mechanism, reagents, operational steps, instrumentation, limitations, sample preparation, and applications in peptide/protein analysis. Questions are built to deepen conceptual understanding and problem-solving skills required for sequencing strategy, troubleshooting common issues (blocked N-termini, disulfide bonds, modified residues), and comparing Edman sequencing with modern mass spectrometry approaches. Use these multiple-choice questions to strengthen your ability to design experiments, interpret sequencing results, and apply appropriate preparatory modifications for reliable N-terminal protein analysis.

Q1. What is the primary reagent used to derivatize the free N‑terminal amino group in Edman degradation?

• Phenylisothiocyanate (PITC)
• Trifluoroacetic acid (TFA)
• Hydroxylamine
• 2,4‑Dinitrofluorobenzene (DNFB)

Correct Answer: Phenylisothiocyanate (PITC)

Q2. Which sequence of steps correctly describes a single Edman degradation cycle?

• Acid cleavage of peptide → PITC coupling under alkaline conditions
• PITC coupling under alkaline conditions → acid cleavage to release PTH‑amino acid
• Oxidation of N‑terminus → enzymatic cleavage
• Alkylation of thiols → PITC coupling

Correct Answer: PITC coupling under alkaline conditions → acid cleavage to release PTH‑amino acid

Q3. What chemical species is produced and identified as the released residue in each Edman cycle?

• Free amino acid
• Phenylthiohydantoin (PTH) derivative of the amino acid
• Carboxyamidomethylated residue
• Peptidyl‑tRNA analogue

Correct Answer: Phenylthiohydantoin (PTH) derivative of the amino acid

Q4. Which modification at the protein N‑terminus most directly prevents Edman degradation from proceeding?

• Disulfide bond formation nearby
• N‑terminal acetylation (blocked N‑terminus)
• Phosphorylation of a serine residue within the sequence
• Methylation of an internal lysine

Correct Answer: N‑terminal acetylation (blocked N‑terminus)

Q5. Practically, Edman degradation reliably sequences how many residues from the N‑terminus before signal loss becomes problematic?

• Only 1–5 residues
• About 10–15 residues
• About 30–50 residues
• More than 200 residues

Correct Answer: About 30–50 residues

Q6. What analytical technique is most commonly used to identify and quantify the released PTH‑amino acids after each cycle?

• Gas chromatography–mass spectrometry (GC‑MS)
• High‑performance liquid chromatography (HPLC) with UV detection
• Capillary electrophoresis without derivatization
• Direct amino acid analysis by ninhydrin colorimetry

Correct Answer: High‑performance liquid chromatography (HPLC) with UV detection

Q7. How does the presence of proline at the N‑terminus affect Edman degradation?

• It completely blocks sequencing; no product forms
• It causes reduced coupling efficiency because proline is a secondary amine
• It increases the yield of PTH‑derivative due to ring stabilization
• It converts PTH‑product into an unstable imide that cannot be detected

Correct Answer: It causes reduced coupling efficiency because proline is a secondary amine

Q8. Which reagent or condition is typically used to cleave the phenylthiocarbamyl derivative and release the PTH‑amino acid?

• Anhydrous trifluoroacetic acid (TFA) or strong acid cleavage
• Hydrogen peroxide oxidation
• Trypsin digestion at 37 °C

Correct Answer: Anhydrous trifluoroacetic acid (TFA) or strong acid cleavage

Q9. For peptides rich in disulfide bonds, what preparative step is usually required before Edman sequencing?

• Oxidize disulfides to sulfonic acids
• Reduce disulfides and alkylate the resulting thiols
• Leave disulfides intact because they aid sequencing
• Glycosylate the cysteines to stabilize them

Correct Answer: Reduce disulfides and alkylate the resulting thiols

Q10. Why is cysteine often chemically modified (e.g., S‑carboxamidomethylation) prior to Edman degradation?

• Because unmodified cysteine produces a PTH derivative that co‑elutes with tryptophan
• Because free thiol groups can form side reactions that interfere with PITC chemistry
• Because cysteine lacks a free alpha‑amino group
• Because cysteine blocks the HPLC column permanently

Correct Answer: Because free thiol groups can form side reactions that interfere with PITC chemistry

Q11. How are PTH‑amino acids typically identified in an automated Edman sequencer?

• By matching mass spectra against a database
• By comparing HPLC retention times and UV spectra to PTH standards
• By measuring radioactivity of separated peaks
• By thin‑layer chromatography only

Correct Answer: By comparing HPLC retention times and UV spectra to PTH standards

Q12. Which statement best contrasts Edman degradation with tandem mass spectrometry for protein sequencing?

• Edman can sequence internal regions without digestion while MS cannot
• Edman provides stepwise N‑terminal sequence information; MS provides mass‑based identification and internal sequence information after fragmentation
• MS cannot detect post‑translational modifications while Edman can
• Edman is generally faster and more high‑throughput than modern MS methods

Correct Answer: Edman provides stepwise N‑terminal sequence information; MS provides mass‑based identification and internal sequence information after fragmentation

Q13. What is the primary cause of decreasing signal intensity with successive Edman cycles?

• Volatilization of the peptide during the assay
• Cumulative loss from incomplete coupling and cleavage efficiencies each cycle
• Enzymatic degradation of the peptide by contaminating proteases
• Progressive oxidation of phenylalanine residues

Correct Answer: Cumulative loss from incomplete coupling and cleavage efficiencies each cycle

Q14. During the PITC coupling step, what is the chemical role of the mildly alkaline environment?

• To protonate the carboxyl terminus so it leaves
• To deprotonate the alpha‑amino group, increasing its nucleophilicity toward PITC
• To oxidize cysteine residues selectively
• To hydrolyze peptide bonds selectively

Correct Answer: To deprotonate the alpha‑amino group, increasing its nucleophilicity toward PITC

Q15. Which functional group on the first residue reacts directly with phenylisothiocyanate in Edman degradation?

• The side‑chain carboxyl group
• The free alpha‑amino group at the N‑terminus
• The C‑terminal carboxyl group
• The peptide backbone carbonyl oxygen

Correct Answer: The free alpha‑amino group at the N‑terminus

Q16. Can standard Edman degradation distinguish between L‑ and D‑amino acids at the N‑terminus?

• Yes — it directly determines absolute stereochemistry
• No — standard Edman sequencing does not distinguish stereochemistry without additional chiral analysis
• Yes — D‑amino acids cannot form PTH derivatives
• No — Edman converts all residues to the L‑form during derivatization

Correct Answer: No — standard Edman sequencing does not distinguish stereochemistry without additional chiral analysis

Q17. What is a typical per‑cycle chemical yield range for Edman reactions in a well‑optimized automated sequencer?

• About 50–60% per cycle
• About 70–80% per cycle
• About 95–99% per cycle
• About 100% per cycle (quantitative)

Correct Answer: About 95–99% per cycle

Q18. Which application is least appropriate for Edman degradation?

• Determination of the first 20–40 N‑terminal residues of a peptide
• Confirming the N‑terminal sequence of a recombinant protein
• Sequencing very large intact proteins (>500 amino acids) without prior cleavage
• Identifying blocked N‑termini by showing lack of PTH release

Correct Answer: Sequencing very large intact proteins (>500 amino acids) without prior cleavage

Q19. After the acid cleavage step in Edman degradation, what is the state of the remaining peptide?

• It is completely hydrolyzed into free amino acids
• It is one residue shorter with a new free N‑terminus ready for the next cycle
• It becomes cyclic and cannot be sequenced further
• It is permanently modified at the C‑terminus

Correct Answer: It is one residue shorter with a new free N‑terminus ready for the next cycle

Q20. If a protein’s N‑terminus is blocked, what common strategy enables Edman sequencing of its primary structure?

• Use mass spectrometry exclusively; Edman is impossible
• Proteolytically digest the protein into peptides and perform Edman on new unblocked N‑termini
• Convert all amino acids to their methyl esters to unblock the terminus
• Sequence the protein from the C‑terminus instead using Edman chemistry

Correct Answer: Proteolytically digest the protein into peptides and perform Edman on new unblocked N‑termini

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