Attomole-level protein fingerprinting based on intrinsic peptide fluorescence

Analytical Chemistry
E Okerberg, J B Shear

Abstract

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.

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Citations

Feb 5, 2002·Electrophoresis·C PrataF Couderc
Nov 30, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Yan HeEdward S Yeung
Apr 1, 2006·Drug Discovery Today. Technologies·Norberto A GuzmanTerry M Phillips
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Dec 1, 2006·Electrophoresis·Ana M García-CampañaHuguette Fabre
Feb 1, 2002·The Journal of Physical Chemistry. a·Ignacy GryczynskiJoseph R Lakowicz
Jun 1, 2005·Analytical Chemistry·Uchenna P PaulPaul B Farnsworth
Jul 2, 2002·Analytical Chemistry·Shen Hu, Norman J Dovichi
Jul 13, 2021·Analytica Chimica Acta·Hai Yen TaFrancois Couderc
Feb 15, 2008·Journal of the American Chemical Society·Andrew J BoydstonChristopher W Bielawski

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