LC-MS in proteomics MCQs With Answer

Introduction:

LC-MS in proteomics is an essential analytical platform in M.Pharm curricula, combining liquid chromatography (LC) for peptide separation with mass spectrometry (MS) for mass measurement and sequencing. This blog-style MCQ set focuses on core concepts and practical aspects of LC-MS workflows used in proteomics research and drug development: sample preparation, chromatographic choices (including nanoLC), ionization methods, mass analyzers, fragmentation techniques, quantitative approaches (label-free and labeled), targeted and discovery strategies, and data analysis concepts like database searching and false discovery rate. The questions are designed to test and deepen understanding for M.Pharm students preparing for exams or research involving modern bio-analytical techniques.

Q1. Which fundamental advantage does coupling liquid chromatography (LC) to mass spectrometry (MS) provide in proteomics?

• It enables real-time visualization of intact proteins without separation
• It separates complex peptide mixtures chromatographically before mass analysis, improving identification sensitivity
• It replaces the need for peptide digestion by measuring intact protein structures directly
• It eliminates the requirement for ionization by detecting neutral molecules

Correct Answer: It separates complex peptide mixtures chromatographically before mass analysis, improving identification sensitivity

Q2. Which ionization technique is most commonly used for on-line LC-MS coupling of proteomic peptide samples?

• MALDI (Matrix-Assisted Laser Desorption/Ionization)
• ESI (Electrospray Ionization)
• Fast Atom Bombardment (FAB)
• Atmospheric Pressure Photoionization (APPI)

Correct Answer: ESI (Electrospray Ionization)

Q3. In LC-MS proteomics, which chromatographic mode is most widely used for peptide separations prior to MS analysis?

• Size-exclusion chromatography
• Ion-exchange chromatography
• Reverse-phase chromatography (often at nano-flow)
• Normal-phase chromatography with nonpolar solvents

Correct Answer: Reverse-phase chromatography (often at nano-flow)

Q4. What is the main purpose of tandem mass spectrometry (MS/MS) in shotgun proteomics?

• To increase chromatographic resolution by adding another separation stage
• To fragment selected precursor ions to obtain sequence-informative fragment ions for peptide identification
• To convert multiply charged ions into singly charged species
• To measure UV absorbance of peptides post-ionization

Correct Answer: To fragment selected precursor ions to obtain sequence-informative fragment ions for peptide identification

Q5. Which mass analyzer is known for very high resolving power and high mass accuracy and is commonly used in proteomics?

• Quadrupole (single)
• Ion trap (3D)
• Orbitrap
• Magnetic sector

Correct Answer: Orbitrap

Q6. Which fragmentation technique is preferred for preserving labile post-translational modifications (e.g., phosphorylation) during peptide fragmentation?

• CID (Collision-Induced Dissociation)
• HCD (Higher-energy Collisional Dissociation)
• ETD (Electron Transfer Dissociation)
• ECD (Electron Capture Dissociation) is never used for peptides

Correct Answer: ETD (Electron Transfer Dissociation)

Q7. Which quantitation strategy involves metabolic incorporation of stable isotope-labeled amino acids into cell proteins?

• iTRAQ (isobaric tags for relative and absolute quantitation)
• TMT (Tandem Mass Tags)
• SILAC (Stable Isotope Labeling by Amino acids in Cell culture)
• Label-free spectral counting

Correct Answer: SILAC (Stable Isotope Labeling by Amino acids in Cell culture)

Q8. Isobaric labeling methods such as iTRAQ and TMT allow multiplexing because they:

• Change peptide retention times so labeled samples elute separately
• Produce reporter ions of different masses in MS/MS that quantify relative abundances
• Convert peptides to DNA tags for sequencing-based quantitation
• Only label proteins at N-termini preventing peptide fragmentation

Correct Answer: Produce reporter ions of different masses in MS/MS that quantify relative abundances

Q9. Which targeted proteomics method uses a triple quadrupole mass spectrometer to monitor specific precursor → fragment ion transitions?

• Shotgun DDA (Data-Dependent Acquisition)
• SRM/MRM (Selected/Multiple Reaction Monitoring)
• PRM (Parallel Reaction Monitoring)
• DIA (Data-Independent Acquisition)

Correct Answer: SRM/MRM (Selected/Multiple Reaction Monitoring)

Q10. In data-dependent acquisition (DDA) configured for shotgun proteomics, how are precursor ions typically chosen for MS/MS?

• All ions within the full m/z range are fragmented simultaneously
• Precursor ions are selected randomly without regard to intensity
• The most intense N precursor ions from each survey scan are selected for MS/MS (top-N)
• Only singly charged ions are targeted for fragmentation

Correct Answer: The most intense N precursor ions from each survey scan are selected for MS/MS (top-N)

Q11. Which approach is commonly used to estimate false discovery rate (FDR) in peptide identifications from MS/MS database searches?

• Using only high-resolution MS data and ignoring low-resolution spectra
• Target-decoy search strategy comparing matches to real and reversed/decoy sequences
• Assuming all identifications with score > 0 are correct without validation
• Counting the number of unique peptides per protein directly

Correct Answer: Target-decoy search strategy comparing matches to real and reversed/decoy sequences

Q12. Mass measurement error is frequently reported in which units in high-resolution proteomics?

• Daltons per second
• Parts per million (ppm)
• Percent relative intensity
• m/z units per minute

Correct Answer: Parts per million (ppm)

Q13. Electrospray ionization (ESI) often produces multiply charged peptide ions. What must be performed to obtain the neutral monoisotopic peptide mass from such spectra?

• Isotopic labeling of the peptide N-terminus
• Deconvolution of charge states to calculate the neutral monoisotopic mass
• Re-running the sample by MALDI to produce singly charged ions
• Only measuring the highest m/z isotope has to be sufficient

Correct Answer: Deconvolution of charge states to calculate the neutral monoisotopic mass

Q14. What is the purpose of applying dynamic exclusion during DDA LC-MS/MS runs?

• To permanently exclude low-abundance peptides from analysis
• To prevent repeated selection of the same precursor ions for MS/MS over a short time, increasing proteome coverage
• To exclude ions below a certain m/z threshold only
• To exclude modifications such as phosphorylation from identification

Correct Answer: To prevent repeated selection of the same precursor ions for MS/MS over a short time, increasing proteome coverage

Q15. Which enrichment method is commonly used to selectively isolate phosphopeptides prior to LC-MS analysis?

• Size-exclusion chromatography
• TiO2 (Titanium dioxide) or IMAC (Immobilized Metal Affinity Chromatography)
• Reverse-phase desalting only
• Hydrophobic interaction chromatography (HIC)

Correct Answer: TiO2 (Titanium dioxide) or IMAC (Immobilized Metal Affinity Chromatography)

Q16. Which database search algorithm historically uses cross-correlation (XCorr) scoring for peptide-spectrum matches?

• Mascot
• SEQUEST
• X! Tandem
• MaxQuant

Correct Answer: SEQUEST

Q17. Peptide mass fingerprinting (PMF) is most commonly associated with which ionization/analyzer combination?

• ESI coupled to Orbitrap with LC online
• MALDI-TOF for mass measurement of intact peptide mixtures
• Triple quadrupole SRM for targeted quantitation
• FT-ICR after gas-phase digestion

Correct Answer: MALDI-TOF for mass measurement of intact peptide mixtures

Q18. Compared to time-of-flight (TOF) analyzers, an Orbitrap typically offers which advantage for proteomic applications?

• Lower mass accuracy but faster acquisition
• Higher resolving power and better mass accuracy
• Exclusive ability to perform CID fragmentation
• Ability to analyze only singly charged ions

Correct Answer: Higher resolving power and better mass accuracy

Q19. How does PRM (Parallel Reaction Monitoring) differ from SRM/MRM in targeted proteomics?

• PRM monitors only precursor ions while SRM monitors only fragments
• PRM acquires high-resolution full MS/MS spectra of chosen precursors, allowing all fragment ions to be measured in parallel, whereas SRM monitors predefined fragment transitions on a triple quadrupole
• PRM requires isotopic labels while SRM does not
• PRM is only used for discovery proteomics, not targeted assays

Correct Answer: PRM acquires high-resolution full MS/MS spectra of chosen precursors, allowing all fragment ions to be measured in parallel, whereas SRM monitors predefined fragment transitions on a triple quadrupole

Q20. What is the function of a lock mass or internal calibrant in high-accuracy LC-MS proteomics workflows?

• To act as an ionization enhancer that increases signal for all peptides
• To provide a constant reference mass that corrects for small mass drifts and improves mass accuracy across a run
• To intentionally shift retention times for better alignment
• To remove background noise by chemical reaction with contaminants

Correct Answer: To provide a constant reference mass that corrects for small mass drifts and improves mass accuracy across a run

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