Protocol to quantify polyphosphate in human cell lines using a tagged PPBD peptide.

Polyphosphate (polyP) is an evolutionarily conserved polymer of phosphates that is difficult to study in human cells because of its low concentration and high lability. First, we described how to express and purify Xpress-tagged PPBD (Ppx1 PolyP Binding Domain). We describe the detection and quantification of nuclear polyP in HEK293T cells using Xpress-PPBD, Xpress antibody, and Alexa-conjugated secondary antibodies. We have also used this protocol in SH-SY5Y HeLa and HEK293 cells. For complete details on the use and execution of this protocol, please refer to Samper-Martín et al. (2021).

Phenylalanine Photoinduced Fluorescence and Characterization of the Photoproducts by LC-MS.

Phenylalanine (Phe) is a direct precursor of tyrosine and several neurotransmitters. The accumulation of Phe in the brain generates serious and not recoverable pathologies in children. Early detection in newborns is fundamental to apply the appropriate therapy and avoid irreversible health problems. Although fluorescence is a sensitive and selective technique for the determination of amino acids, the fluorescent analysis of Phe is limited since it exhibits a very low fluorescence quantum yield; however, the fluorescence of Phe increases drastically under UV irradiation when a peroxide medium is used. The aim of this research was to analyze the effect of the UV-radiation on Phe aqueous-peroxide solutions and to study the influence of the chemical environment on the photoinducted fluorescence process. The nature and characteristics of the fluorescent photoproducts generated under off-line UV irradiation in hydrogen peroxide medium were achieved by high performance liquid chromatography (HPLC) using a spectrophotometer detector (DAD) coupled in series with a mass spectrometer (MS) or with a fast scan spectrofluorimetric detector (FSFD). Environmental characteristics such as pH, initial concentration of Phe, hydrogen peroxide amount and irradiation time were studied in order to establish their influence on the formation of each one of the photoproducts. As the formation of several highly fluorescent photoproducts has been confirmed, the possibility of designing a chromatographic system with a post-column on-line photoreactor is open. The measure of the total fluorescence signal generated from Phe at the optimized irradiation time, could be used for the determination, with high sensitivity, of the initial amount of Phe in aqueous media, such as human serum or environmental samples. These aspects are being studied at present. .