Quantitative amino acid analysis of subnanogram levels of protein by open tubular liquid chromatography.

A method is described for the hydrolysis and quantitative amino acid analysis of as little as 0.1 ng (4 fmol) of protein. Hydrolysis is performed by using a gas-phase method. The resulting amino acids are derivatized with naphthalene-2,3-dicarboxaldehyde and then analyzed by open tubular liquid chromatography with amperometric detection. The total volume present after derivatization is approximately 25 nL. To our knowledge this is currently the lowest level of protein to be quantitatively analyzed, as well as the smallest volume in which derivatization has been accomplished. It was possible to quantitatively determine 14 amino acids with an error of 5.0% for known protein and 8.5% for a protein whose identity was unknown to the researcher.