The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones

Inositol phosphates (InsPs) are ubiquitous in all eukaryotes. However, since there are 63 possible different phosphate ester isomers, the analysis of InsPs is challenging. In particular, InsP1, InsP2, and InsP3 already amass 41 different isomers, of which some occur as enantiomers. Profiling of these “lower” inositol phosphates in mammalian tissues requires powerful analytical methods and reference compounds. Here, we report an analysis of InsP2 and InsP3 with capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS). Using this method, the bacterial effector RipBL1 was analyzed and found to degrade InsP6 to Ins(1,2,3)P3, an understudied InsP3 isomer. This new reference molecule then aided us in the assignment of the isomeric identity of an InsP3 while profiling human samples: in urine and kidney stones, we describe for the first time the presence of defined and abundant InsP3 isomers, namely Ins(1,2,3)P3, Ins(1,2,6)P3 and/or Ins(2,3,4)P3.


RipBL1 expression and purification
For expression, an overnight culture of Escherichia coli BL21-CodonPlus (DE3)-RIL cells (Stratagene, La Jolla, CA, USA) harboring pDest527-RipBL1 was inoculated in 2YT containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol. At OD 600 = 0.8, the culture was cooled on ice and protein expression was induced by 400 µM IPTG and continued shaking at 16°C for 22 hours.
To purify RipBL1, the cell pellet was resuspended in 300 mM NaCl, 25 mM Na 2 HPO 4, pH 7.5, 2 mM DTT, 1 mM EDTA and 1 mg/ml lysozyme. After 30 min at 4°C, rotating, cells were disrupted by vortexing for 1 min with glass beads, followed by incubation on ice for 1 min, each step repeated eight times. The cell lysate was collected by centrifugation at 2800 g for 15 min, followed by centrifugation at 18000 g for 45 min at 4°C, respectively. The lysate was incubated with Ni-NTA-resin (Protino) overnight and then washed in 300 mM NaCl, 25 mM Na 2 HPO 4, pH 7.5, 25 mM imidazole and 1 mM DTT. The protein was eluted in the same buffer but with 250 mM imidazole. Protein purity and concentration of dialysed RipBL1 was checked by SDS-PAGE and Coomassie staining and BSA standards.

Extraction of PP-InsPs and InsPs from kidney stone and urine
Urine samples were obtained from fresh void of the second morning urine and immediately frozen at -80°C upon further processing. Nine kidney stone formers and 10 controls free of stone (no kidney stone history and no detectable calcification on an abdominal CT-scan) from the Swiss Kidney Stone Cohort (SKSC) did participate, the characteristic of the participants are shown in Table S1. SKSC is registered at ClinicalTrial.gov under the identifier NCT01990027 and was approved by the Swiss ethic committee. Extraction of kidney stones: kidney stones (composition determined by IR spectroscopy) were powdered with a grinder, then 10 mg powder was weighted and homogenized in 1 ml of ice-cold perchloric acid (1M), incubated at 4°C with rotation for 20 minutes. The homogenate was centrifuged with max speed. The supernatant was transferred to pre-washed titanium dioxide (TiO 2 ) beads for InsPs purification. [1] Briefly, the supernatant was incubated with 5 mg TiO 2 beads at 4°C for 20 minutes to capture inositol phosphates. The beads were washed with 1M perchloric acid solution and then the inositol phosphates eluted with 3% ammonium hydroxide. The elution solution was dried in a speed vac evaporator.
Extraction of urine: centrifuge 0.4 ml urine at 2000g at 4°C for 5 minutes. Transfer the supernatant to 0.4 ml of 2 M perchloric acid (ice-cold) and rotate at 4°C for 20 minutes. The homogenate was centrifuged with max speed. Transfer the supernatant to 10 mg of prewashed titanium dioxide (TiO 2 ) beads for InsPs purification as described above.

CE-QQQ analysis
All kidney stone extract samples were dissolved in 50 μl MilliQ water, and all urine extracts were dissolved in 30 μl MilliQ water. Quantitation of InsP 5 and InsP 6 were performed with known amounts of isotopic standards spiked as internal standards.  Figure S1. Calibration curves for quantitation of Ins (1,2) P 2 and Ins (1,2,6) P 3 with standards.
Area of peaks are plotted against the concentration of standards. A signal-to-noise ratio between 3 to 10 is considered acceptable for the LOD, and a signal-to-noise ratio above 10 is considered acceptable for the LOQ.

Figure S10
High-resolution mass spectra of InsPs from CE-qTOF analysis of urine from a