Characterization of a benzyladenine binding-site peptide isolated from a wheat cytokinin-binding protein: sequence analysis and identification of a single affinity-labeled histidine residue by mass spectrometry

• Published in: Proceedings of the National Academy of Sciences - PNAS 1988-08, Vol.85 (16), p.5927-5931
• Main Authors: Brinegar, A.C, Cooper, G, Stevens, A, Hauer, C.R, Shabanowitz, J, Hunt, D.F, Fox, J.E
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; 6-BENZILADENINA; 6-BENZYLADENINE; Adenine - analogs & derivatives; Adenine - metabolism; ADENINES; Affinity Labels; AMINES; AMINO ACID SEQUENCE; AMINO ACIDS; ANTIMETABOLITES; Arabidopsis Proteins; AROMATICS; AZAARENES; AZOLES; BASIC BIOLOGICAL SCIENCES; Benzyl Compounds; Binding Sites; CARBON 14 COMPOUNDS; CARBOXYLIC ACIDS; Carrier Proteins - analysis; CEREALS; CHEMICAL REACTIONS; CHROMATOGRAPHY; CITOCININAS; CROSS-LINKING; cytokinin; CYTOKININE; CYTOKININS; DRUGS; EMBRIONES VEGETALES; EMBRYON VEGETAL; ESPECTROSCOPIA DE MASAS; GRAMINEAE; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; HISTIDINA; HISTIDINE; Histidine - analysis; IMIDAZOLES; ION EXCHANGE CHROMATOGRAPHY; Irradiation; Kinetin; KININS; LABELLED COMPOUNDS; LIGANDS; LILIOPSIDA; MAGNOLIOPHYTA; Mass spectra; Mass Spectrometry; MASS SPECTROSCOPY; MOLECULAR STRUCTURE; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PEPTIDE; PEPTIDES; PEPTIDOS; Photolysis; PLANT EMBRYOS; Plant Proteins; PLANTS; POLYMERIZATION; POLYPEPTIDES; PROTEINAS; PROTEINE; PROTEINS; PURINES; Radioactive decay; SEPARATION PROCESSES; Sequencing; Solvents; SPECTROGRAPHIE DE MASSE; spectrometry; SPECTROSCOPY; TRITICUM; Triticum durum; WHEAT
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.16.5927

A wheat embryo cytokinin-binding protein was covalently modified with the radiolabeled photoaffinity ligand 2-azido-N6-[14C]benzyladenine. A single labeled peptide was obtained after proteolytic digestion and isolation by reversed-phase and anion-exchange HPLC. Sequencing by classical Edman degradation identified 11 of the 12 residues but failed to identify the labeled amino acid. Analysis by laser photodissociation Fourier-transform mass spectrometry of 10 pmol of the peptide independently confirmed the Edman data and also demonstrated that the histidine residue nearest the C terminus (underlined) was modified by the reagent in the sequence Ala-Phe-Leu-Gln-Pro-Ser-His-$\underline{\text{His}}$-Asp-Ala -Asp-Glu.