A proteome-scale study on in vivo protein Nα-acetylation using an optimized method

Area of Science:

• Proteomics
• Post-translational modifications
• Biochemistry

Background:

• Protein N-terminal acetylation (N(α)-acetylation) is a prevalent eukaryotic modification.
• Previous enrichment methods using CNBr-activated Sepharose resin had limitations, including potential loss of Lys-containing peptides and the need for sample-to-resin ratio optimization.

Purpose of the Study:

• To develop an optimized method for enriching N(α)-modified peptides.
• To overcome limitations of previous enrichment techniques, enhancing efficiency and scope.
• To expand the dataset of identified acetylated N-termini.

Main Methods:

• Introduction of a double incubation step at pH 6.0 for peptide enrichment.
• Inclusion of singly charged precursors for MS/MS fragmentation.
• Analysis of 80 μg samples in duplicate using the optimized method.

Main Results:

• The optimized method successfully enriches N(α)-modified peptides irrespective of ε-NH(2) presence, removing the need for ratio optimization.
• Identification of 922 IPI annotated and 103 IPI unannotated acetylated N-termini from 989 proteins, establishing the largest dataset to date.
• Discovery of unexpected coupling of CNBr-activated Sepharose to His-containing peptides.

Conclusions:

• The optimized double incubation method significantly improves N-terminal acetylation enrichment.
• This approach provides a more robust and comprehensive analysis of the N(α)-acetylome.
• The study highlights the reproducibility and complementarity of the N(α)-acetyl proteome approach.