We constructed a novel QIT-TOF mass spectrometer. We obtained room temperature UV PD spectra at the home of singly protonated tyrosine and singly protonated hexapeptides, DYYVVR. DYYVVR is a tryptic peptide of Janus kinase 3 and has the two active tyrosine residues needed to regulate of the protein’s activity by phosphorylation. Although we obtained a well-resolved spectrum at room temperature for singly protonated tyrosine, two adjacent tyrosine absorption spectra in singly protonated DYYVVR were not distinguishable. Therefore, cryogenic ion spectroscopy (CIS) was used to elucidate the spectrum results of singly protonated DYYVVR at room temperature. Through comparison with point mutants, it was determined that the lower electronic band to the absorption of 981st tyrosine, while the higher band to 980th tyrosine. In the case of phosphorylation, the UV absorption of the phosphorylated chromophore shifts to higher energy above 36500 cm–1. In contrast, the unphosphorylated chromophore retains its absorption in the same region. It is proved that CIS can be used to identify the two adjacent tyrosine chromophores and to confirm the phosphorylation site of a kinase homology domain. In the case of room temperature, UVPD spectra are also used to differentiate the absorption of two adjacent tyrosine chromophores. Mass spectrometry and ultraviolet spectroscopy in 2-dimensional analysis can effectively demonstrate to identify the location of phosphorylation sites in a peptide.
