Statistical validation of 1H NMR protocol vs standard biochemical assay in quality control of RBC packed units.

BACKGROUND: Time dependent quantification of endogenous metabolites in biological samples (blood, urine, biological tissues extracts) in normal and pathological conditions as well as following therapeutic protocols is well established. In the clinical practice, such a dynamic flux of information allows the physician to identify and appreciate alterations associated to biochemical pathways of specific organs. In the years, many biochemical assays have been developed to detect, selectively, this vast array of molecules. METHODS: The Proton Nuclear Magnetic Resonance (1H NMR) spectrum allows the identification and quantification of more than 30 RBC-associated metabolites with minimum manipulation of the sample. To validate the use of 1H NMR spectroscopy for quality control purposes in transfusion medicine, a series of statistical tools have been employed to analyse and compare accuracy and precision of the 1H NMR results with respect to the ones obtained by standard biochemical assays. RESULTS: Among the many metabolites that can be detected and quantified by 1H NMR spectroscopy we selected creatinine and lactate, since they are routinely quantified by standard biochemical assays and because they are characterized by a wide concentration dynamic range. We show that 1D 1H NMR spectroscopy is an accurate a precise method for metabolite quantification. CONCLUSION: These results validate the use of 1H NMR spectroscopy in transfusion medicine as a method to evaluate the quality of RBC packed units and to develop novel and more efficient RBCs storage protocols.

Development of a strategy for the total chemical synthesis of an allergenic protein: the peach LTP Pru p 3.

The possibility to obtain allergenic proteins by means of total chemical synthesis would be a big step forward in the development of cures to food allergy and in the study of the mechanism of allergic reactions, because this would allow to achieve control at the molecular level over the structure of the product and to study its relationship with the allergenic activity in fine details. This is instead not possible by using allergens produced by extraction from natural sources or by recombinant DNA techniques. In this work, we aimed to test for the first time the feasibility of the total chemical synthesis of an allergenic protein. Pru p 3, the most studied member of the family of lipid transfer proteins, relevant plant food pan-allergens, was used as model target. Strategies for the convergent assembly of the target protein, starting from five peptide fragments to be bound by means of either native chemical ligation or peptide hydrazide ligation, followed by desulfurization, to achieve ligations at alanine, were developed and tested. All the reaction conditions were set up and optimized. Two large peptides covering the two halves of the protein sequence were synthesized and structurally characterized by means of circular dichroism, and their immunogenicity was proved by means of immunoblot, using antibodies against Pru p 3, and immunoCAP inhibition tests. Finally, the five peptides were bound together to produce the whole protein stretch. The obtained results demonstrate the feasibility of total chemical synthesis as a new way to obtain pure allergens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.