Question

Most accurate method to determine the molecular weight of a given polypeptide is:

• A. Gel permeation chromatography
• B. SDS-PAGE
• C. Analytical ultracentrifugation
• D. MALDI-TOF mass spectrometry

Hint:

MALDI-TOF mass spectrometry is considered the gold standard for determining the molecular weight of polypeptides because of its high precision and speed.

Answer 1

The Correct Option is D

Three of these four methods estimate molecular weight indirectly, through comparison with reference standards or physical behaviour under a force, rather than measuring mass directly.
Gel permeation chromatography and SDS-PAGE both estimate size relative to marker standards, introducing approximation.
Analytical ultracentrifugation infers weight from sedimentation behaviour, useful but less precise for a single accurate figure.
MALDI-TOF mass spectrometry directly measures the mass of ionised molecules from their flight time, giving the most accurate molecular weight of the four.
So the correct answer is MALDI-TOF mass spectrometry.

Answer 2

Grouping these four techniques by what physical property they actually measure, rather than what they are commonly used for, clarifies which one gives the most direct and accurate molecular weight figure.

1. Gel permeation chromatography: This technique measures elution volume through a column, which correlates with size but depends heavily on the shape and hydration of the molecule, making it an indirect and less precise estimate of mass.
2. SDS-PAGE: This technique measures migration distance in an electric field, which correlates with size under denaturing conditions but again is calibrated against markers rather than measuring actual mass.
3. Analytical ultracentrifugation: This technique measures the rate of sedimentation under a strong centrifugal field, from which molecular weight can be calculated, but the calculation depends on assumptions about density and shape that add uncertainty.
4. MALDI-TOF mass spectrometry: This technique measures the mass-to-charge ratio of ionised molecules directly through their time of flight, a property tied fundamentally to mass itself rather than to an indirect physical behaviour, which is why it delivers the highest accuracy.

Only one of the four techniques measures mass as a direct physical quantity rather than inferring it from an indirect property, and that is MALDI-TOF.

Therefore, the correct answer is MALDI-TOF mass spectrometry.