The chemistry of protein sequence analysis

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• N-terminal sequence analysis using Edman chemistry is crucial for protein and peptide structural determination.
• Current methods primarily rely on enhanced sensitivity of on-line analysis of phenylthiohydantoin (PTH) amino acids via reverse-phase high-performance liquid chromatography (RP-HPLC).

Purpose of the Study:

• To review and critically evaluate alternative Edman reagents and chemistries, including fluorescent methods, for increasing protein sequencing sensitivity.
• To discuss instrumental constraints and challenges hindering the routine application of advanced sequencing techniques.

Main Methods:

• Review of existing literature on Edman chemistry and its advancements.
• Critical evaluation of alternative reagents, fluorescent chemistries, and chromatographic methods (e.g., microbore columns).
• Analysis of instrumental limitations impacting sequencing sensitivity.

Main Results:

• Sensitivity improvements in Edman sequencing have reached the 1-5 picomole (pmol) range using microbore RP-HPLC columns.
• Alternative reagents and fluorescent chemistries show promise for femtomole-level sequencing but are not yet in routine use.
• Current chromatographic methods face limitations, suggesting that further sensitivity gains solely through this approach are unlikely.

Conclusions:

• Advancing the sensitivity of N-terminal sequencing necessitates further research into both novel chemical approaches and improved instrumentation.
• Alternative separation and detection methodologies may be essential for achieving significantly higher sensitivity in protein and peptide sequencing.
• Overcoming current limitations requires a multidisciplinary approach integrating chemistry and engineering for future Edman chemistry developments.