Determining protein primary structure — complementary strategies Which of the following strategies are used together to determine the sequence of amino acids (primary structure) of a protein?

Introduction / Context:
Protein sequencing integrates chemical and enzymatic methods to reconstruct the order of amino acids. Classic workflows combine end-group analysis, selective cleavage, chromatographic separation, and fragment sequencing (Edman degradation or tandem mass spectrometry). Understanding why multiple complementary steps are required ensures reliable reconstruction of the full sequence, including disulfide mapping.

Given Data / Assumptions:

• Proteins are often too large to sequence intact by one method.
• Terminal residue identification constrains sequence assembly.
• Fragmentation at defined sites (e.g., trypsin, CNBr) generates overlapping peptides.

Concept / Approach:

Terminal analyses (N- and C-termini) provide anchors for assembling fragments. Controlled cleavage yields overlapping sets that can be aligned to deduce order. Modern tandem MS adds speed and sensitivity but still benefits from strategic proteolysis and knowledge of termini. Therefore, a comprehensive approach uses all listed strategies rather than relying on a single technique in isolation.

Step-by-Step Solution:

Verification / Alternative check:

Concordance between independent fragment maps and termini assignments validates the final sequence; discrepancies trigger re-analysis for modifications or proteolysis artifacts.

Why Other Options Are Wrong:

Choosing only one technique risks ambiguities. Mass spectrometry alone (E) is powerful but typically paired with digestion and database/terminal constraints for de novo cases.

Common Pitfalls:

Ignoring post-translational modifications, disulfide bonds, or N-terminal blockage that require tailored strategies (e.g., reduction/alkylation, enzymatic deglycosylation).

Final Answer:

All of the above