High-sensitivity amino acid analysis by derivatization with O-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection: applications in protein structure determination.
An amino acid analysis method using a commercially available analyzer that accurately quantitates protein-derived amino acids in the 10-100 pmol range is described. The method utilizes the robotic capability of the analyzer's autosampler to perform precolumn derivatization of both primary and secondary amino acids with o-phthalaldehyde and 9-fluorenylmethyl chloroformate, respectively. The derivatized amino acids are then separated on a C-18 reverse-phase amino acid column and quantitated in a single run by fluorescence detection. The characterization of beta-lactoglobulin and two tryptic peptides from the bacterial enzyme diaminopimelic acid epimerase is used to demonstrate the sensitivity and utility of this method.
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Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Krivanek
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Ackermann
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Cardin
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D000596:Amino Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D005449:Fluorenes; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D009842:Oligopeptides / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D015717:Peptide T; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D009764:o-Phthalaldehyde
D000596:Amino Acids; D005449:Fluorenes; D007202:Indicators and Reagents; D009842:Oligopeptides; D010446:Peptide Fragments; C053805:peptide T (4-8); D015717:Peptide T; D009764:o-Phthalaldehyde; C054007:1-(9-fluorenyl)methyl chloroformate