抄録
Capillary high-performance liquid chromatography was connected on-line to the electrospray interface of a quadrupole mass spectrometer. Samples containing dilute protein or nucleic acids in salt-containing buffers were desalted and concentrated in the capillary reversed-phase column, and the column eluate was directly analyzed with the electrospray mass spectrometry. Both intact proteins purified from bovine brain and peptide mixtures made from the proteins by protease digestion were successfully analyzed by the LC/MS method. With two phosphoproteins known as in vivo major substrates of protein kinase C, various species with a mass difference of 80 Da were resolved, suggesting that the isolated proteins contained species phosphorylated in vivo to different degrees. Protease digests were analyzed with the same LC/MS method to identify the in vivo phosphorylation sites. The phosphopeptides thus identified were further subjected to MS/MS analysis to establish exact phosphorylation sites. The same methodology was applied to studies on nucleic acids, especially on the posttranscriptional modifications of tRNA. The results thus obtained revealed many hitherto unknown sites of modifications in proteins and in nucleic acids, demonstrating the power of the LC/MS analysis based on electrospray ionization.
