High sensitivity detection of tryptic digests using derivatization and fluorescence detection

Abstract

Publisher Summary Derivatization of peptide mixtures with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) has been shown to an effective means of generating useful peptide maps from protein tryptic digests. This chapter describes the application of this procedure to tryptic digests of a model protein that discusses useful maps with subpicomole amounts of sample. The choice of HPLC column and fluorescent detector flow cell can be optimized to provide subpicomole detection, but there is some compromise between operating conditions that provide the maximum sensitivity and the highest resolution. Both standard peptide maps and derivatized maps are readily generated from a single digest, which provides additional information for a complex peptide mixture without requiring significantly more sample. However, several key features of this derivatization analysis are still not fully explored, and volatile buffer systems, such as the ammonium bicarbonate that is briefly discussed, will permit the facile collection of fractions for further analysis, such as amino acid composition or mass spectrometry.