Selective Labeling Of Proteins On Their N-Termini With Synthetic Peptides

Background

Direct and selective labeling of α-amines or α-carboxylates of the N-terminal amino acid in proteins is a powerful approach for profiling proteins in complex mixtures. Approximately 80% of mammalian proteins are N-terminally acetylated. Thus, N-terminal labeling provides greater signal over background than does C-terminal labeling. However, such labeling must still be extremely selective for α-amines over lysine ε-amines, which are approximately 25 times more abundant in an average protein.

Technology Description

Investigators at UCSF have invented a general method to selectively label proteins on their N-termini with synthetic peptides. Briefly, this method uses an engineered enzyme subtiligase and a peptide substrate specially tailored to the proteomic workflow. The substrate comprises a peptide glycolate ester with a subtiligase cleavage site, a biotin label, and a TEV-cleavage site. The action of the subtiligase enzyme results in proteins that are selectively biotinylated on the N-terminus. The biotin label can then be used to capture these labeled proteins from a complex mixture using immobilized avidin. Finally, the TEV cleavage site allows for the enrichment of captured proteins or peptides for downstream analysis such as mass spectroscopy. Other N-terminal labels can also be used in place of biotin making the process very versatile.             

Application

• Specific labeling of α-amines of N-terminal amino acid in proteins

• Global proteomic profiling of complex mixtures of proteins and polypeptides. 
  

Advantages

• High specificity for the N-terminus of proteins 
• Selective labeling of α-amines over lysine ε-amines of proteins      

Stage of Development

Pre-clinical

Related Materials

• Mahrus S, et al. Global Sequencing of Proteolytic Cleavage Sites in Apoptosis by Speci?c Labeling of Protein N Termini. Cell 134, 866–876, September 5, 2008
• Wildes, D & Wells, J.A. (2010) Proc. Natl. Acad. Sci. USA 107(10), 4561-4566. “Sampling the N-terminal proteome of human blood”. PMID: 20173099
• Agard, N.J, et al (2010) Mol. Cell. Proteomics 5, 880-893. “Inflammatory stimuli regulate caspase substrate profiles”. PMID: 20173201
• Agard, N.J., et al (2012) Proc Natl Acad Sci U S A. 109:1913-8. “Global kinetic analysis of proteolysis via quantitative targeted proteomics”. PMID: 22308409
• Crawford, E.D., Seaman, J.E., et al (2012), “The DegraBase: A database of proteolysis in healthy and apoptotic human cells”, Mol Cell Proteomics

Patent Status