Attomole-level protein fingerprinting based on intrinsic peptide fluorescence

Protein identification has relied heavily on proteolytic analysis, but curr ent techniques are often slow and generally consume large quantities of val uable protein sample. We report the development of a rapid, ultralow volume protein analysis strategy based on tryptic digestion within the tip of a 1 .5-mum capillary channel followed by separation of the proteolytic fragment s using capillary electrophoresis (CE). Two-photon excitation is used to pr obe the intrinsic fluorescence of peptide fragments through "deep-UV" excit ation of aromatic amino acid residues at the outlet of the CE channel. Dete ction limits using this technique are 0.7, 2.4, and 23 amol for the aromati c amino acids tryptophan, tyrosine, and phenylalanine, respectively. In the se studies, we demonstrate the capacity to differentiate bovine and yeast c ytochrome c variants using less than 15 amol of protein through tryptic fin gerprinting, Moreover, the detection of a single amino acid substitution be tween bovine and canine cytochrome c illustrates the sensitivity of this ap proach to minor differences in protein sequence. The 2-pL sample volume req uired for this on-column tryptic digestion is, to our knowledge, the smalle st yet reported for a proteolytic assay.