A proteomics approach to study in vivo protein N(alpha)-modifications

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• N-terminal modifications play crucial roles in protein function and regulation.
• Existing methods for identifying N-terminal modifications are often limited in scope and efficiency.

Purpose of the Study:

• To develop and validate a simple method for enriching and identifying peptides with in vivo N(alpha)-modified protein N-termini.
• To characterize the types and prevalence of N-terminal modifications in HeLa cells.

Main Methods:

• Utilized CNBr-activated Sepharose to selectively couple peptides via the alpha-NH(2) group under mild conditions.
• Applied LC-MS/MS analysis to tryptic and Lys-N digests of HeLa cell proteins following enrichment.
• Investigated and discussed methods for removing artifactual N(alpha)-modifications.

Main Results:

• Identified 588 in vivo N(alpha)-modified peptides from tryptic digests, with 507 from IPI-annotated N-termini.
• Identified 394 in vivo N(alpha)-modified peptides from Lys-N digests, with 371 from IPI-annotated N-termini.
• Combined analysis revealed 675 N(alpha)-modified IPI-annotated and 88 N(alpha)-modified IPI-unannotated protein N-termini, predominantly acetylations.

Conclusions:

• The developed method efficiently enriches and identifies N(alpha)-modified peptides.
• Results suggest N-terminal acetyltransferases (NATs) may also possess N-terminal formyltransferase (NFT) and N-terminal propionyltransferase (NPT) activities in vivo.