Mass spectrometric top-down analysis of proteins

Area of Science:

• Proteomics and Mass Spectrometry
• Biopharmaceutical Analysis

Background:

• Traditional mass spectrometry (MS) methods, involving enzymatic digestion, offer limited protein sequence coverage (20-80%).
• This limitation is insufficient for comprehensive characterization, particularly for biopharmaceuticals, risking loss of critical sequence information.

Purpose of the Study:

• To introduce and detail the top-down sequencing approach for comprehensive protein analysis.
• To highlight the advantages of top-down sequencing over traditional bottom-up methods for detailed protein characterization.

Main Methods:

• Top-down sequencing analyzes intact proteins, starting with intact mass determination.
• Intact proteins are fragmented within the mass spectrometer to generate fragment ions.
• Fragment ions are analyzed to determine protein sequence, modifications, and mutations.

Main Results:

• Top-down sequencing provides complete amino acid sequence information and identifies post-translational modifications.
• This approach ensures no loss of sequence information, offering higher accuracy than bottom-up strategies.
• Recent technological advancements have expanded the capabilities of top-down sequencing.

Conclusions:

• Top-down sequencing is essential for comprehensive protein analysis, especially for biopharmaceuticals.
• Complementary application of top-down and bottom-up strategies on the same instrument yields more detailed protein insights.
• This integrated approach enhances the understanding of protein structure, modifications, and mutations.