Recent advances in organic chemistry enabled organic reactions in aqueous media with high yield under mild condition. This chemical evolution has promoted chemical modification of proteins, which contributes to protein engineering and chemical biology research. Here we demonstrate N-terminal glycine specific labeling of proteins by Pictet-Spengler reaction in combination with transamination reaction using pyridoxal-5-phosphate (PLP) under physiological condition. Horse heart myoglobin, a 153 amino acid residue heme-binding protein containing N-terminal glycine residue was oxidized by PLP, and the resultant aldehyde was coupled with tryptophan analogues in phosphate buffer (pH 6.5) at 37℃ to give corresponding 1,2,3,4-tetrahydro-β-carboline derivatives. Achromobacter protease I (Lys-C) liberated peptides were purified by ODS-HPLC to give N-terminal peptide (K1-peptide; the region from residue 1 to K16), which was analyzed by MALDI postsource decay (PSD) fragmentation. The ion peaks observed in the MALDI-PSD spectrum were assigned along the K1-peptide containing β-carboline skeleton, not 1,2,3,4-tetrahydro-β-carboline skeleton. A possible reason for this phenomenon is that the 1,2,3,4-tetrahydro-β-carboline skeleton might be spontaneously dehydrated during HPLC purification and laser desorption ionization. The circular dichroism (CD) spectral profiles of he modified myoglobin and the wild-type myoglobin were superimposable, which result showed that the tertiary structure of myoglobin was not altered during the reaction. Furthermore, modification of myoglobin was confirmed by SDS-PAGE and Western blot with no sign of decomposition.
