Isotope labeling in proteomics MCQs With Answer

Isotope labeling in proteomics MCQs With Answer

This quiz set is designed for M.Pharm students studying proteins and protein formulations, focusing on isotope labeling strategies used in proteomics. It covers principles, commonly used labeling methods (SILAC, TMT, iTRAQ, ICAT, 18O), metabolic versus chemical labeling, MS1- versus MS2-based quantification, practical considerations (labeling efficiency, multiplexing, ratio compression), and applications like turnover studies and absolute quantification. Questions mix conceptual understanding and applied problem-solving relevant to experimental design, data interpretation, and troubleshooting in quantitative proteomics. Answers are provided to help self-assess and deepen comprehension for lab practice and exam preparation.

Q1. What is the main principle behind SILAC (Stable Isotope Labeling by Amino acids in Cell culture)?

• Chemical tagging of peptides after digestion with heavy isotopes
• Metabolic incorporation of heavy isotope-labeled amino acids into proteins during cell culture
• Enzymatic exchange of oxygen atoms to incorporate 18O into peptides
• Affinity enrichment of cysteine-containing peptides using isotopic probes

Correct Answer: Metabolic incorporation of heavy isotope-labeled amino acids into proteins during cell culture

Q2. Which labeling strategy produces reporter ions in MS2 spectra for relative quantification across multiple samples?

• SILAC
• TMT (Tandem Mass Tag)
• 18O labeling
• Label-free quantification

Correct Answer: TMT (Tandem Mass Tag)

Q3. In 18O labeling of peptides, where do the isotopes incorporate?

• On lysine side chains during translation
• Into the peptide C-terminus during protease-catalyzed hydrolysis
• At methionine residues by oxidation
• Into peptide backbone nitrogen atoms

Correct Answer: Into the peptide C-terminus during protease-catalyzed hydrolysis

Q4. Which statement best describes iTRAQ labeling?

• Metabolic labeling of whole organisms with 13C amino acids
• Chemical isobaric labeling of peptides at the N-terminus and lysine side chains producing reporter ions in MS2
• Stable isotope labeling by enzymatic 18O incorporation only for intact proteins
• Affinity-based capture of labeled cysteine residues for absolute quantification

Correct Answer: Chemical isobaric labeling of peptides at the N-terminus and lysine side chains producing reporter ions in MS2

Q5. What is “ratio compression” commonly observed with isobaric tags like TMT/iTRAQ?

• Underestimation of true fold changes caused by co-isolated contaminant peptides contributing to reporter ion signals
• Overestimation of peptide mass due to isotopic impurities
• Loss of peptide identifications after labeling due to digestion inefficiency
• Increased dynamic range of quantification leading to exaggerated ratios

Correct Answer: Underestimation of true fold changes caused by co-isolated contaminant peptides contributing to reporter ion signals

Q6. Which labeling method is most appropriate for studying protein turnover rates in cultured cells?

• SILAC (pulse-SILAC) metabolic labeling
• TMT 10-plex labeling
• ICAT targeting cysteines
• Label-free spectral counting

Correct Answer: SILAC (pulse-SILAC) metabolic labeling

Q7. ICAT reagents specifically target which amino acid residue for labeling?

• Lysine
• Arginine
• Cysteine
• Serine

Correct Answer: Cysteine

Q8. Which of the following is an advantage of MS1-based quantification (e.g., SILAC) over MS2-based isobaric quantification?

• Higher multiplexing capacity (>10 samples) by default
• Less susceptible to ratio compression from co-isolated peptides
• Generates reporter ions for easy comparison in MS2
• Does not require high mass accuracy instruments

Correct Answer: Less susceptible to ratio compression from co-isolated peptides

Q9. Multiplexing capacity refers to:

• The number of peptides identified per run
• The number of different samples that can be compared simultaneously using a labeling method
• The dynamic range of the mass spectrometer
• The efficiency of enzymatic digestion

Correct Answer: The number of different samples that can be compared simultaneously using a labeling method

Q10. Why is isotopic purity important in isotope labeling experiments?

• It determines the efficiency of enzymatic digestion
• Impure isotopes can create overlapping isotopic envelopes and bias quantification accuracy
• It affects peptide solubility in LC buffers
• It only matters for label-free methods

Correct Answer: Impure isotopes can create overlapping isotopic envelopes and bias quantification accuracy

Q11. Which approach provides absolute quantification of a target protein rather than relative quantification?

• Label-free intensity-based comparison
• Using isotopically labeled synthetic peptides as internal standards (AQUA/SIS)
• iTRAQ multiplex relative comparison
• Pulse-SILAC without internal standards

Correct Answer: Using isotopically labeled synthetic peptides as internal standards (AQUA/SIS)

Q12. A major practical limitation of metabolic labeling (e.g., SILAC) is:

• Incompatibility with cultured cells
• Requires live cell growth and cannot be applied easily to tissues or body fluids
• Generates reporter ions that are interfered with by contaminants
• Cannot label arginine residues

Correct Answer: Requires live cell growth and cannot be applied easily to tissues or body fluids

Q13. Which MS acquisition strategy can help reduce ratio compression for TMT experiments?

• Using lower resolution MS1 scans
• Performing MS3 (SPS-MS3) to isolate reporter ions after additional fragmentation
• Skipping chromatographic separation to speed acquisition
• Using only CID fragmentation at low collision energy

Correct Answer: Performing MS3 (SPS-MS3) to isolate reporter ions after additional fragmentation

Q14. Chemical labeling after digestion (e.g., iTRAQ/TMT) commonly labels which functional groups?

• Carboxyl groups only
• Amine groups at peptide N-termini and lysine side chains
• Sulfhydryl groups on cysteines exclusively
• Phosphate groups on phosphopeptides

Correct Answer: Amine groups at peptide N-termini and lysine side chains

Q15. In SILAC experiments, incomplete incorporation of heavy amino acids leads to:

• Improved signal-to-noise because light peptides vanish
• Complex isotopic envelopes and inaccurate quantification due to mixed populations
• No effect as MS detects only labeled species
• Conversion of lysine to arginine leading to peptide loss

Correct Answer: Complex isotopic envelopes and inaccurate quantification due to mixed populations

Q16. Which labeling strategy uses isotopic exchange with H2(18)O during proteolysis to introduce mass shifts?

• ICAT
• 18O labeling
• SILAC
• TMT

Correct Answer: 18O labeling

Q17. For targeted quantification of low-abundance proteins, which isotope-based approach is commonly used with SRM/MRM?

• Pulse-SILAC metabolic labeling without standards
• Use of stable isotope-labeled peptide standards (AQUA) spiked into the sample
• Global TMT multiplexing for discovery
• ICAT for cysteine depletion only

Correct Answer: Use of stable isotope-labeled peptide standards (AQUA) spiked into the sample

Q18. Which factor can cause quantification errors when using isobaric tags on modified peptides (e.g., phosphopeptides)?

• Phosphorylation prevents MS detection altogether
• Variable enrichment efficiency and altered fragmentation can change reporter ion yield and bias ratios
• Isobaric tags cannot label modified peptides
• Phosphopeptides always produce more intense reporter ions than unmodified peptides

Correct Answer: Variable enrichment efficiency and altered fragmentation can change reporter ion yield and bias ratios

Q19. Which is a benefit of metabolic labeling over chemical labeling in terms of sample handling?

• Requires more complex chemical reactions after digestion
• Labels are introduced in vivo, avoiding extra chemical modification steps during sample prep
• Allows labeling of peptides from archived tissue samples directly
• Always provides higher multiplexing than chemical tags

Correct Answer: Labels are introduced in vivo, avoiding extra chemical modification steps during sample prep

Q20. When designing an experiment using isobaric tags, which control is most important to detect labeling or mixing artifacts?

• Omitting protease digestion in one sample
• Including a pooled reference or common internal standard across all channels
• Using different LC gradients for each channel
• Only running each labeled sample separately without mixing

Correct Answer: Including a pooled reference or common internal standard across all channels

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