Methods of protein sequencing MCQs With Answer

Methods of protein sequencing MCQs With Answer

This quiz collection focuses on experimental and analytical methods used to determine protein primary structure, tailored for M.Pharm students. It covers classical chemical approaches such as Edman degradation and Sanger’s reagent, enzymatic and chemical cleavage strategies (trypsin, CNBr), sample preparation (reduction/alkylation, blocking issues), and modern mass spectrometry techniques including MALDI-TOF, ESI-MS/MS, peptide mass fingerprinting and de novo sequencing. Emphasis is placed on interpretation of fragmentation patterns (b/y ions), identification of post-translational modifications, and practical limitations when sequencing large or modified proteins. Each MCQ challenges applied understanding to prepare students for laboratory decision-making and data analysis in protein characterization.

Q1. Which reagent is used in Edman degradation to derivatize the N-terminal amino acid prior to its cleavage and identification?

• Phenylisothiocyanate (PITC)
• 2,4-Dinitrofluorobenzene (FDNB)
• Dansyl chloride
• Cyanogen bromide (CNBr)

Correct Answer: Phenylisothiocyanate (PITC)

Q2. Which limitation most directly prevents Edman degradation from sequencing a protein?

• Presence of disulfide bonds
• Blocked N-terminus (e.g., acetylation)
• High molecular weight above 50 kDa
• Multiple methionine residues

Correct Answer: Blocked N-terminus (e.g., acetylation)

Q3. Cyanogen bromide (CNBr) cleavage is specific for which peptide bond?

• C-terminal side of methionine residues
• N-terminal side of tryptophan residues
• C-terminal side of lysine and arginine residues
• N-terminal side of cysteine residues

Correct Answer: C-terminal side of methionine residues

Q4. Which enzyme is most commonly used to generate peptides by cleavage at the C-terminal side of lysine and arginine for MS-based sequencing?

• Trypsin
• Chymotrypsin
• Carboxypeptidase A
• Pepsin

Correct Answer: Trypsin

Q5. In tandem mass spectrometry (MS/MS) peptide fragmentation, which ion series corresponds to fragments that include the N-terminus?

• b-ions
• y-ions
• a-ions
• z-ions

Correct Answer: b-ions

Q6. Peptide mass fingerprinting (PMF) using MALDI-TOF compares experimentally observed peptide masses to predicted peptide masses from database digests. What is a primary limitation of PMF?

• It cannot distinguish isobaric amino acid substitutions (e.g., Leu/Ile)
• It requires radioactive labeling of peptides
• It provides detailed site-specific PTM localization
• It inherently sequences intact proteins without digestion

Correct Answer: It cannot distinguish isobaric amino acid substitutions (e.g., Leu/Ile)

Q7. Which chemical reagent is traditionally used in Sanger’s method to label the free N-terminal amino acid for identification?

• 2,4-Dinitrofluorobenzene (FDNB)
• Phenylisothiocyanate (PITC)
• Iodoacetamide
• Dansyl chloride

Correct Answer: 2,4-Dinitrofluorobenzene (FDNB)

Q8. In sample preparation for sequencing, why is reduction and alkylation of cysteine residues performed?

• To prevent reformation of disulfide bonds and stabilize cysteines for analysis
• To cleave peptide bonds adjacent to cysteines
• To remove N-terminal acetylation blocking groups
• To selectively label glycosylation sites

Correct Answer: To prevent reformation of disulfide bonds and stabilize cysteines for analysis

Q9. Which mass spectrometry ionization technique is typically paired with liquid chromatography and is useful for sequencing peptides by MS/MS?

• Electrospray ionization (ESI)
• Fast atom bombardment (FAB)
• Electron impact ionization (EI)
• MALDI-TOF without LC

Correct Answer: Electrospray ionization (ESI)

Q10. Which statement best describes “de novo” peptide sequencing by tandem MS?

• Inferring peptide sequence directly from MS/MS fragment ion masses without database matching
• Matching peptide masses against a protein database to identify proteins
• Using Edman chemistry to read residues sequentially
• Cleaving proteins exclusively with CNBr prior to analysis

Correct Answer: Inferring peptide sequence directly from MS/MS fragment ion masses without database matching

Q11. Which modification commonly blocks Edman degradation and requires alternative strategies for sequencing?

• N-terminal acetylation
• Carbamidomethylation of cysteine
• Oxidation of methionine
• Phosphorylation of serine

Correct Answer: N-terminal acetylation

Q12. Which analytical output from Edman sequencing is typically detected and identified by HPLC as a derivative?

• Phenylthiohydantoin (PTH)-amino acids
• Dansyl-amino acids
• 2,4-Dinitrophenyl (DNP)-amino acids
• Carboxymethylated cysteines

Correct Answer: Phenylthiohydantoin (PTH)-amino acids

Q13. When interpreting MS/MS spectra, which pair of fragment ions differ by 18 Da and indicate a neutral loss of water?

• a-ion and b-ion pairs with water loss
• b-ion and b-ion-H2O variants
• y-ion and a-ion differences
• z-ion and y-ion differences

Correct Answer: b-ion and b-ion-H2O variants

Q14. Which strategy is most appropriate to determine the sequence of a large (>100 kDa) protein?

• Proteolytic digestion into peptides followed by MS/MS or peptide sequencing
• Direct Edman sequencing of the intact 100 kDa molecule
• Using CNBr alone without further fragmentation or analysis
• Measuring intact molecular weight by MALDI-TOF and inferring sequence

Correct Answer: Proteolytic digestion into peptides followed by MS/MS or peptide sequencing

Q15. Which database search algorithm is commonly used to identify proteins from MS/MS data by matching observed spectra to theoretical peptides?

• Mascot
• BLAST
• ClustalW
• EdmanPro

Correct Answer: Mascot

Q16. Which reagent is used to alkylate reduced cysteines to prevent re-formation of disulfide bonds and create a stable mass shift for MS analysis?

• Iodoacetamide (carbamidomethylation)
• Phenylisothiocyanate (PITC)
• 2,4-Dinitrofluorobenzene (FDNB)
• Cyanogen bromide (CNBr)

Correct Answer: Iodoacetamide (carbamidomethylation)

Q17. Which fragmentation pathway in CID (collision-induced dissociation) is most commonly observed for peptide backbone cleavage to give sequence-informative ions?

• Cleavage of the amide bond producing b- and y-ions
• Cleavage of the disulfide bond producing c- and z-ions
• Side-chain neutral losses only, without backbone cleavage
• Preferential N-terminal fragmentation producing x-ions

Correct Answer: Cleavage of the amide bond producing b- and y-ions

Q18. For localization of phosphorylation sites on a peptide, which MS approach is most useful?

• Tandem MS/MS with targeted fragmentation and site-determining ions
• Intact mass measurement by MALDI-TOF without fragmentation
• Edman degradation of the intact phosphorylated protein
• CNBr cleavage followed by UV absorbance at 280 nm

Correct Answer: Tandem MS/MS with targeted fragmentation and site-determining ions

Q19. Which method can be used to map disulfide bond connectivity in a protein?

• Partial reduction, alkylation labeling, proteolytic digestion and MS analysis of linked peptides
• Direct application of Edman degradation to intact, oxidized protein
• Peptide mass fingerprinting without reduction
• Gel electrophoresis with Coomassie staining only

Correct Answer: Partial reduction, alkylation labeling, proteolytic digestion and MS analysis of linked peptides

Q20. Which analytical technique allows separation and sequencing of peptides produced from enzymatic digestion prior to MS or Edman sequencing, improving sequence coverage?

• High-performance liquid chromatography (HPLC)
• Simple centrifugation
• Paper chromatography without detectors
• Direct UV absorbance of whole protein

Correct Answer: High-performance liquid chromatography (HPLC)

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