Exopeptidase Assisted N- and C-Terminal Proteome Sequencing

Area of Science:

• Proteomics and Molecular Biology
• Biochemistry and Biophysics

Background:

• In vivo proteoforms can differ from genomic sequences due to post-translational modifications.
• Understanding protein N- and C-termini is crucial for elucidating protein functions.
• Current methods for terminal sequencing are limited to single proteins or require complex enrichment strategies for proteome-wide analysis.

Purpose of the Study:

• To present a novel, unbiased method for ladder sequencing of protein N- and C-termini.
• To overcome the limitations of existing proteome-wide terminal identification techniques.
• To enable comprehensive characterization of protein termini across entire proteomes.

Main Methods:

• Combined exopeptidase digestion of the proteome with two-dimensional chromatographic separation.
• Utilized tandem mass spectrometry (MS/MS) for sensitive detection and sequencing.
• Applied the methodology to analyze the N- and C-terminome of *Saccharomyces cerevisiae*.

Main Results:

• Successfully identified 2190 N-termini and 1562 C-termini in *S. cerevisiae*.
• Demonstrated the unbiased and comprehensive nature of the developed sequencing approach.
• Validated the method's potential for large-scale proteome analysis.

Conclusions:

• The novel method significantly expands the proteomics toolbox for terminal protein characterization.
• Enables sequential analysis of N- and C-termini for entire proteomes.
• Provides a powerful new tool for understanding protein diversity and function.