Fluorescent isotope-coded affinity tag (FCAT). I: Design and synthesis.
A novel class of isotope-coded affinity tag is proposed possessing a fluorescent feature, referred to as fluorescent isotope-coded affinity tag (FCAT), to provide a new tool for quantitative proteomics. The label is designed to bind cysteine containing proteins or peptides. The FCAT reagent comprises four functional elements: a specific chemical reactivity group toward sulfhydryl groups; a linker that can incorporate the stable isotopes; a hydroxymethylbenzoic residue (base labile group) to cleave off a large part of the label before MS analysis; and a fluorescent tag for absolute quantification. The fluorescent part of the tag is also planned to be utilized to isolate the FCAT-labeled peptides via antibody based pull-down method. In this paper, we report on the solid phase organic synthesis of the light isotope containing FCAT molecule. The new labeling reagent showed good reactivity with model cysteine containing peptides. The fluorophore group was also effectively cleaved off from the labeled products to accommodate easier MS based analysis.
Horváth Laboratory of Bioseparation Sciences, Institute of Analytical Chemistry and Radiochemistry, University of Innsbruck, Innrain 52a, Innsbruck A-6020, Austria.
Zuly
Z
Rivera-Monroy
Guenther K
GK
Bonn
András
A
Guttman
D019817:Benzoic Acid / Q000737:chemistry; D002621:Chemistry / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D007202:Indicators and Reagents / Q000138:chemical synthesis / Q000737:chemistry; D007554:Isotopes; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D008968:Molecular Conformation; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007554:Isotopes; D010455:Peptides; D013438:Sulfhydryl Compounds; D019817:Benzoic Acid; D003545:Cysteine