Background

Inositol phosphates (IPs) are a family of conserved signaling molecules, ubiquitous in eukaryotes (Irvine and Schell, 2001; Tsui and York, 2010). They have been implicated in the regulation of a broad range of cellular activities, including calcium signaling, trafficking, and phosphate homeostasis (Wilson et al., 2013; Thota and Bhandari, 2015; Azevedo and Saiardi, 2017). However, our understanding of IPs signaling has been hampered by the fact that they can be difficult to study.

Unlike other phosphate-rich molecules such as nucleotides, IPs do not absorb in the UV/Vis range, and are often present at relatively low abundance in cells. The traditional methodology for IP detection and analysis is to radioactively metabolically label cells with ³H-inositol that is taken up and incorporated into IPs over 1-5 days (Wilson and Saiardi, 2017). After labeling, IPs are extracted with perchloric acid; these extracts are neutralised before separation by strong anion exchange (SAX) HPLC and scintillation counting of each fraction (Azevedo and Saiardi, 2006). The use of ³H-labelled IPs and chromatography has also been required for in vitro biochemical studies. The requirement for radioactive IPs or metabolic labeling limits the possible lines of investigation. These are time-consuming, technically demanding and expensive experiments.

We previously developed a polyacrylamide gel electrophoresis (PAGE) method for resolving and visualizing IPs (Losito et al., 2009; Loss et al., 2011). This technique was immediately useful in following in vitro reactions, as well as analyzing in vivo high abundance IPs such as IP₆, IP₇ and IP₈ from Dictyostelium discoideum (Pisani et al., 2014), or IP₆ from plant seeds (Desai et al., 2014; Kolozsvari et al., 2014). However, in the majority of cell types or model organisms, low IP concentrations make it impossible to run a PAGE gel of enough neutralized extract to visualize IPs while maintaining correct gel migration. In mammalian cells, the most abundant IP is IP₆ at 40-100 µM (measured in cell lines such as HL60, C1866 and BAF3; French et al., 1991; Bunce et al., 1993), while the inositol pyrophosphate IP₇ is thought to be present at sub-µM levels. We were therefore inspired to develop the present method that uses titanium dioxide beads to purify cold or radioactive IPs regardless of their abundance (Wilson et al., 2015). Titanium dioxide binds the phosphate groups of the IPs. The concentrated IPs can be analyzed by PAGE, SAX-HPLC, or other techniques.

The use of titanium dioxide beads now enables analysis of total unlabeled IPs from any cell type (Pavlovic et al., 2015; Wilson et al., 2015; Gu et al., 2016; Pavlovic et al., 2016). It also allows the study of IPs extracted from previously impossible sample types, including large volumes of liquid media, biofluids, or animal tissues. For biochemical work, the method can be used to remove salt and proteins from IPs preparations. Here we present the method as used for purifying IPs from cultured adherent mammalian cells.