Site-specific fluorescent derivatization and liquid chromatographic-mass spectrometric characterization of long R(3) IGF-I for bioanalytical applications.
Recombinant Long R(3) IGF-I was derivatized with fluorescein isothiocyanate (FITC) at a single location by careful selection of reaction conditions (i.e. pH, and FITC/protein amino group ratio). High-performance liquid chromatography (LC) and electrospray mass spectrometry (MS) were used to confirm the extent of fluorescein conjugation. The protein conjugate was isolated and subjected to cyanogen bromide (CNBr) cleavage, followed by LC-MS to determine the site of modification. The isolated species of Long R(3) IGF-I-FITC was labeled at the N-terminal Met residue. Recognition of this fluorescent analog by monoclonal anti-IGF-I was preserved, indicating its potential for immunodiagnostic applications.
Harvey
Michael D
MD
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard W, Montreal, QC, H3G 1M8, Canada
Banks
Peter R
PR
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007334:Insulin-Like Growth Factor I / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data
D005456:Fluorescent Dyes; D007334:Insulin-Like Growth Factor I