Fluorescence-labeled peptides as isoelectric point (pI) markers in capillary isoelectric focusing with fluorescence detection.
Commercially available peptides, mostly angiotensin derivatives, were labeled at their N-terminal amino group with 5-carboxytetramethylrhodamine succinimidyl ester, to obtain fluorescent pI markers for capillary isoelectric focusing with fluorescence detection. The labeled peptides were purified by reversed-phase chromatography. They were well separated on isoelectric focusing in a polyacrylamide gel slab and their pIs were determined by comigration with protein-pI markers. The fluorescent markers could be detected as sharp peaks in capillary isoelectric focusing with laser-induced fluorescence detection (He-Ne laser, 1 mW, 543.5 nm). The detection limit was found to be around 3 x 10(-12) M (0.8 amol). Tetramethylrhodamine-labeled pea lectin (3 pg) was subjected to capillary isoelectric focusing and the pIs of the fluorescent derivatives of the lectin were determined by using the fluorescence-labeled peptides as pI markers.
Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
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Shimura
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Kasai
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D037102:Lectins / Q000737:chemistry; D008433:Mathematics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D037121:Plant Lectins; D012235:Rhodamines; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D037102:Lectins; D010455:Peptides; D037121:Plant Lectins; D012235:Rhodamines; C036292:pea lectin; C005358:tetramethylrhodamine