MoP-23


AMINO ACID ANALYSIS BY GAS CHROMATOGRAPHY AND ECNI MASS SPECTROMETRY

Anne Cerpa-Poljak1 and Mark W. Duncan1,2

1CRC for Biopharmaceutical Research, Sydney
2BMSU, University of NSW, Sydney


Amino acid analysis (AAA) is a useful aid in protein chemistry, but its routine application is limited by poor precision, accuracy, and a modest limit-of-detection, when compared with other protein chemistry tools. Typically, 10 pmol of material is required to provide meaningful data, although some groups report being able to work with as little as 1 pmol of material. Additionally, when low pmoles of protein are analysed the reproducibility can be poor and significant errors in amino acid composition usually arise. AAA is therefore usually employed in preference to Edman sequencing only where large amounts of material are available (>100 pmol), or where proteins are N-terminally blocked.

In the work described here we have utilised stable isotope dilution and combined gas chromatography - electron-capture negative ionisation (ECNI) mass spectrometry to obtain improvements in accuracy, sensitivity and analysis time. This approach yields reliable data on less than 100 fmoles of material. All 20 protein amino acids have been determined in this manner, with an analysis time of ca. 20 min. A mixture of 20 stable isotope labelled amino acids are added to the protein, which is then hydrolysed in the conventional manner using HCl. The hydrolysate is derivatised using an adaptation of the method of Hayashi et al (1986) to form stable, volatile, electron-capturing derivatives which are injected onto the GC-MS system. The ratio of the ion current for the selected ions for the native amino acid and its stable isotope labelled form is directly proportional to the amounts of the two compounds in the sample.

Hayashi et al, J. Chromatogr., 380 (1986) 239-245