Tags for labeling protein N-termini with subtiligase for proteomics

We explored whether it would be possible to improve the yield of the tagging reaction and increase proteomic coverage by producing more soluble peptide esters

Hikari A.I. Yoshihara; Sami Mahrus; James A. Wells

2008

Scholarcy highlights

  • The peptide ligase subtiligase, derived from subtilisin, has been employed in the identification of protein N-termini in complex mixtures
  • A complex mixture of proteins is N-terminally labeled with subtiligase using a peptide ester containing a biotin affinity tag and a TEV protease cleavage site
  • Tagged N-terminal peptides are captured on avidin beads, cleaved off the beads by TEV protease treatment and analyzed by LC-MS/MS
  • In principle, increasing amount of peptide ester will increase the extent of the ligation reaction by providing more ester for ligation before it is consumed by hydrolysis
  • We explored whether it would be possible to improve the yield of the tagging reaction and increase proteomic coverage by producing more soluble peptide esters
  • Adding propargylglycine residues into the peptide esters allows the quantitiation of the labeled N-termini by sandwich ELISA following derivatization with a nitrotyrosine and azide containing peptide

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