Site-specific fluorescent derivatization and liquid chromatographic-mass spectrometric characterization of long R(3) IGF-I for bioanalytical applications |
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Authors: | Harvey Michael D Banks Peter R |
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Affiliation: | Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard W, Montreal, QC, H3G 1M8, Canada |
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Abstract: | 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. |
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Keywords: | Derivatization, LC Corresponding author. Tel.: +1-514-937-9949 fax: +1-514-937-0007. |
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