RESUMO
Protein phosphorylation is one of the most common and extensively studied post-translational modifications (PTMs). Compared to the O-phosphorylation on Ser, Thr and Tyr residues, our understanding of arginine phosphorylation is relatively limited, both in prokaryotes and eukaryotes, due to the intrinsic instability of phosphoarginine (pArg) and the lack of a feasible method to produce anti-pArg antibodies. We report the design and synthesis of a stable pArg analog, in which the labile N-P bond is replaced with a non-hydrolyzable C-P bond. Significantly, this analog was successfully used as a hapten to raise an immune response and the first mouse polyclonal antibody that specifically recognizes pArg-containing peptides and proteins was produced using analog-KLH conjugated as the immunogen. The generated antibody shows excellent specificity towards pArg-containing peptides and proteins, and could be used for a variety of biological detection methods. This provides us an invaluable tool to unravel the mystery of the biological function of pArg.
Assuntos
Anticorpos/imunologia , Arginina/análogos & derivados , Animais , Anticorpos/química , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Antígenos/química , Antígenos/imunologia , Arginina/síntese química , Arginina/química , Arginina/imunologia , Desenho de Fármacos , Camundongos , Estrutura Molecular , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , Compostos Organofosforados/imunologia , Peptídeos/química , Peptídeos/imunologia , Proteínas/química , Proteínas/imunologiaRESUMO
The identification of carbohydrate isomers, including mono units, linkage positions and anomeric configurations, remains an arduous subject. In this study, the natural amino acid leucine (Leu) was found to specifically interact with cellobiose (Cello) to form a series of potassium adducts as [Cello + Leu + K](+), [Cello + 2Leu + K](+), and [2Cello + Leu + K](+) in the gas phase using mass spectrometry. By using CID-MS/MS, these complexes produced specific fragmentation patterns from the sugar backbone cleavage instead of non-covalent interactions. Moreover, their fragment distributions were dependent on the ratios of Cello-to-Leu in the complexes and the fragmentation pathways of potassium-cationized disaccharides (Dis) were remarkably changed with leucine binding. It should be pointed out that the ternary complex [2Cello + AA + K](+) was unique for leucine among all the twenty natural amino acids. The [2Dis + Leu + K](+) complex produced the most informative fragments by tandem mass spectrometry, which was successfully applied for rapid and efficient discrimination of twelve glucose-containing disaccharide isomers in combination with statistical analyses including PCA and OPLS-DA. The methodology developed here not only provides a novel analytical approach for the differentiation of disaccharide isomers, but also brings new sight towards the interactions of amino acids with disaccharides.