LC-MS and 2D-SDS-PAGE effectively separate proteins of identical molecular weight by leveraging differences in charge, hydrophobicity, or isoelectric point. The correct answer is option (4): B and C only.

Technique Evaluation

SDS-PAGE (A) denatures proteins with SDS, giving uniform negative charge per mass, so separation relies solely on molecular weight through a polyacrylamide gel—ineffective for same-weight proteins.

2D-SDS-PAGE (B) combines isoelectric focusing (first dimension, by pI/charge) with SDS-PAGE (second dimension, by mass); same-mass proteins with different pI separate orthogonally into distinct spots.

Mass spectrometry with liquid chromatography (C) uses LC for initial separation (e.g., reverse-phase by hydrophobicity) followed by MS for precise mass-to-charge analysis; detects sequence/post-translational differences even at identical nominal mass.

Gel filtration (D), or size-exclusion chromatography, separates by hydrodynamic volume (correlates with mass for globular proteins)—fails for identical masses.​

Gel filtration + SDS-PAGE (E) combines two mass-based methods, offering no orthogonal separation.​

Option Breakdown

• (1) E, A Only: Wrong—both mass-dependent, cannot resolve same-weight proteins.

• (2) A, D Only: Wrong—both rely on size/mass alone.

• (3) A, B Only: Wrong—SDS-PAGE (A) fails despite 2D-PAGE (B) succeeding.

• (4) B and C Only: Correct—both provide additional dimensions (pI or hydrophobicity/MS) beyond mass.

Separating two proteins with same molecular weight requires techniques beyond size-based methods like SDS-PAGE or gel filtration, which fail due to identical mass/hydrodynamic volume. Orthogonal approaches like 2D electrophoresis or chromatography-MS succeed by exploiting charge or hydrophobicity differences.

Why Mass-Based Methods Fail

SDS-PAGE coats proteins uniformly with SDS (1.4 g SDS/kDa), migrating solely by length through pores—same MW means co-migration. Gel filtration elutes by exclusion from pores; globular proteins of equal mass elute together.

Effective Techniques

• 2D-SDS-PAGE: First-dimension IEF separates by pI (net charge at pH gradient); second-dimension SDS-PAGE by mass. Spots form at (pI, MW) coordinates—resolves isoforms.

• LC-MS: HPLC (e.g., C18 column) separates by hydrophobicity/sequence; MS provides exact mass (distinguishes isotopes/PTMs). Ideal for complex mixtures.

Exam Strategy

For CSIR NET/NEET, identify “same MW” as cue for non-size methods. Option (4) fits as B/C add charge/MS resolution.