Sanger sequencing cannot be used to determine the sequence of a novel protein.​

The correct answer is (C) Sanger sequencing. This technique sequences DNA, not proteins directly, making it unsuitable for novel proteins without a corresponding gene sequence.​

Option Analysis

(A) De novo sequencing by ESI-MS/MS directly determines amino acid sequences from tandem mass spectra of peptides without database reliance, ideal for novel proteins.​

(B) Edman degradation sequentially removes and identifies N-terminal amino acids from peptides, providing direct protein sequence data up to 50 residues.​

(C) Sanger sequencing relies on chain-terminating dideoxynucleotides to sequence DNA, not proteins; any protein application stems from inferred translation.​

(D) Peptide mass fingerprinting matches peptide masses to databases for identification but fails for novel proteins lacking entries, as it does not generate sequences.​

Protein sequencing techniques enable precise determination of amino acid order in novel proteins, crucial for CSIR NET life sciences preparation. Among options like de novo sequencing by ESI-MS/MS, Edman degradation, Sanger sequencing, and peptide mass fingerprinting, one stands out as unsuitable.​

De Novo Sequencing by ESI-MS/MS

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) fragments peptides and interprets mass spectra to reconstruct sequences ab initio. This method excels for unknown proteins, using algorithms to deduce amino acids from fragment ions.​

• Handles post-translational modifications.

• Independent of databases.

• High-throughput for proteomics.

Edman Degradation Method

This chemical process labels and cleaves N-terminal amino acids iteratively, identifying each via chromatography. Automated sequencers achieve 30-50 residues, perfect for peptide ladders in novel protein analysis.​

• Direct amino acid identification.

• No genome needed.

• Complements mass spectrometry.

Why Sanger Sequencing Fails

Sanger sequencing uses dideoxynucleotides for DNA chain termination, reading nucleotide order via electrophoresis. It indirectly infers protein sequences via translation but cannot directly sequence proteins, especially novel ones without DNA data.​

Peptide Mass Fingerprinting Limitations

PMF digests proteins into peptides, measures masses via MALDI/ESI-MS, and matches to databases. For novel proteins, absent database entries prevent identification or sequencing.​

• Fast for known proteins.

• No sequence output.

• Requires trypsin digestion.