ADVANTAGES AND LIMITATIONS OF DERIVATIZATION OF PEPTIDES FOR IMPROVED PERFORMANCE AND DETECTABILITY IN CAPILLARY ISOELECTRIC FOCUSING (CIEF)

Abstract

Several proteins of varying molecular weights (Mr) were shown to produce a single species, or multiple species which behaved as a single species, upon analysis with capillary isoelectric focusing (cIEF) after derivatization with a large molar excess of the derivatization reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Increased molar excesses of reagent were required as the molecular weight (Mr) of the sample increased. The derivative products exhibited acidic pI shifts, improved peak efficiencies, and lowered (improved) detection limits when compared to the native species. In at least one case, a derivative product (not fully tagged) was shown to exhibit antibody (Ab) recognition when challenged with an Ab, raising the possibility of using these derivatives in affinity recognition studies (e.g., affinity CE, immuno-CE, and so forth). Problems were encountered with precipitation during derivatization and focusing. This problem was more pronounced with the more basic proteins. This would appear to limit the applicability of this reagent as a universal derivatization reagent for use with cIEF studies. The results presented herein represent a promising technique, and they offer advantages as well as certain limitations. Though not yet a perfect approach towards improved analysis and identification of peptides in cIEF, these results indicate tangible opportunities for further optimization.

ACNWOLEDGMENTS

This work was supported, in part, by Zeneca Pharmaceuticals Corporation, Wilmington, DE, through a generous grant from the Drug Disposition and Metabolism Section (N. LeDonne and K. Kirkland). Additional funding, technical materials, and support were provided Northeastern University by Waters Corporation, Milford, MA (U. Neue and R. Pfeifer). Certain cIEF coated capillaries were provided by J&W Scientific, Folsom, CA (D. Mao). The MALDI-TOFMS spectra were obtained through the generous assistance of several individuals within PE Biosystems, Perkin-Elmer Corporation, Framingham, MA (K. Waddell, D. Patterson and S. Martin).