Attomole-level protein fingerprinting based on intrinsic peptide fluorescence.
Protein identification has relied heavily on proteolytic analysis, but current techniques are often slow and generally consume large quantities of valuable 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-microm capillary channel followed by separation of the proteolytic fragments using capillary electrophoresis (CE). Two-photon excitation is used to probe the intrinsic fluorescence of peptide fragments through "deep-UV" excitation of aromatic amino acid residues at the outlet of the CE channel. Detection limits using this technique are 0.7, 2.4, and 23 amol for the aromatic amino acids tryptophan, tyrosine, and phenylalanine, respectively. In these studies, we demonstrate the capacity to differentiate bovine and yeast cytochrome c variants using less than 15 amol of protein through tryptic fingerprinting. Moreover, the detection of a single amino acid substitution between bovine and canine cytochrome c illustrates the sensitivity of this approach to minor differences in protein sequence. The 2-pL sample volume required for this on-column tryptic digestion is, to our knowledge, the smallest yet reported for a proteolytic assay.
Okerberg
E
E
Department of Chemistry & Biochemistry, and the Institute for Cellular & Molecular Biology, University of Texas, Austin 78712, USA.
Shear
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000032:analysis / Q000145:classification; D004285:Dogs; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis*; D012441:Saccharomyces cerevisiae; D014357:Trypsin
D003574:Cytochrome c Group; D010455:Peptides; D014357:Trypsin