High-performance liquid chromatographic peptide mapping and amino acid analysis in the sub-nanomole range

Abstract

By ensuring adequate gradient mixing and eliminating all major artifact peaks we have been able to obtain reproducible high-performance liquid chromatographic tryptic peptide maps on less than 50 pmol of protein. Likewise, a 10–20 fold improvement in the sensitivity of amino acid analysis has been achieved by analyzing the phenylthiocarbamyl derivatives of the free amino acids rather than the free amino acids themselves. This approach enables accurate amino acid compositions to be obtained on less than 50 ng of protein, providing that a simple correction is made for the background level of serine and glycine. We have been able to reduce the background level of these two amino acids to ca. 10 pmol each per sample by incinerating the hydrolysis tubes at 500°C prior to introduction of the sample and by using gas-phase as opposed to liquid-phase hydrolysis. Background corrections are not necessary when over 500 ng of protein are hydrolyzed. With this amount of protein, amino acid compositions, based on phenylthiocarbamyl amino acid analyses were, on average, found to be accurate to within ± 10%.