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

Step 1: Understanding the techniques.
MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry is a powerful technique used for determining the molecular weight of molecules, including polypeptides. It ionizes the sample and measures the time it takes for ions to travel through a flight tube, providing accurate molecular weight measurements.
Step 2: Analyzing the options.
(1) Gel permeation chromatography: This method separates molecules based on size but does not provide precise molecular weight measurements. It is useful for estimating size but not for accurate molecular weight determination.
(2) SDS-PAGE: SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) separates proteins based on their size, but it does not directly measure molecular weight. The results need to be compared to a molecular weight standard.
(3) Analytical ultracentrifugation: This technique measures the sedimentation rate of particles in a centrifugal field but is less commonly used than MALDI-TOF for precise molecular weight determination.
(4) MALDI-TOF mass spectrometry: Correct — MALDI-TOF is the most accurate and widely used method for determining the molecular weight of polypeptides, as it directly measures the molecular ions and provides precise data.
Step 3: Conclusion.
The correct answer is (4) MALDI-TOF mass spectrometry, as it offers the most accurate molecular weight determination for polypeptides.

Answer 2

The question asks for the most accurate method among several established techniques for estimating a polypeptide's molecular weight, so comparing the precision and basis of measurement of each technique identifies the best answer.

1. Gel permeation chromatography: This technique separates molecules by size as they pass through a porous matrix and estimates molecular weight by comparison with reference standards, which introduces error from assumptions about shape and behaviour rather than a direct mass measurement.
2. SDS-PAGE: This technique separates proteins according to their migration distance in a gel relative to standard markers, giving only an approximate molecular weight based on relative mobility rather than a directly measured mass.
3. Analytical ultracentrifugation: This technique estimates molecular weight from sedimentation behaviour under centrifugal force, which is informative for studying protein shape and interactions but is more laborious and less precise for a straightforward molecular weight figure than a direct mass measurement.
4. MALDI-TOF mass spectrometry: This technique ionises the polypeptide and measures the actual time it takes ions to travel a fixed distance, calculating mass directly from that flight time, giving a highly precise, direct measurement of molecular weight rather than an estimate based on migration or sedimentation behaviour.

Comparing a direct mass measurement against three methods that only estimate size or migration shows that MALDI-TOF gives the most accurate result.

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