Inorganic polyphosphate (polyP) is an ancestral, ubiquitous, and well-conserved polymer, which is present in all the studied organisms. It is formed by individual subunits of orthophosphate which are linked together by bonds structurally similar and isoenergetic to those found in ATP. While the metabolism and the physiological roles of polyP in some organisms, including bacteria and yeast, have already been described, the exact role of this polymer in mammalian physiology remains still poorly explored. In these organisms, polyP shows a co-localization with mitochondria. Accordingly, its role as a key regulator of bioenergetics has already been demonstrated by our group and others. Here, using Wild-type (Wt) and MitoPPX (cells enzymatically depleted of mitochondrial polyP) SH-SY5Y cells, we conducted a comprehensive study of the status of cellular physiology, using proteomics and metabolomics approaches. Our results suggested a clear dysregulation of mitochondrial physiology, especially of bioenergetics, in MitoPPX cells when compared with Wt. Interestingly, the effects induced by the enzymatically depletion of polyP are similar to those present in the mitochondrial dysfunction which is observed in neurodegenerative disorders and in neuronal aging. In fact, the role of polyP as a component of the cellular stress responses has already been proposed in diverse organisms, including mammals. Thereafter, our data could contribute to place the metabolism of mitochondrial polyP as a valid and innovative pharmacological target in these conditions.

Cells were resuspended in 100 ul of 5% SDS, 50mM Triethylammonium bicarbonate (TEAB), 2mM MgCl2, 1XHALT phosphatase and protease inhibitors briefly probe sonicated and vortexed. Protein concentration was measured with a BCA assay. Homogenate of 50 ug was added to a process control of 800 ng of yeast enolase protein (Sigma) which was then reduced with 20 mM DTT and alklyated with 40 mM IAA. Lysates were then prepared for S-trap column (Protifi) binding by the addition of 1.2% phosphoric acid and 350 ul of binding buffer (90% Methanol, 100 mM TEAB). The acidified lysate was bound to column incrementally, followed by 3 wash steps with binding buffer to remove SDS and 3 wash steps with 50:50 methanol:chloroform to remove lipids and a final wash step with binding buffer. Trypsin (1:10) in 50mM TEAB was then added to the S-trap column for digestion at 47°C for one hour. Hydrophilic peptides were then eluted with 50 mM TEAB and hydrophobic peptides were eluted with a solution of 50% acetonitrile in 0.2% formic acid. Elutions were pooled, speed vacuumed and resuspended in 0.1% formic acid. A heavy labeled Peptide Retention Time Calibrant (PRTC) mixture (Pierce) was added to each sample. One ug of each sample with 150 femtomole of PRTC was loaded onto a 30 cm fused silica picofrit (New Objective) 75 μm column and 3.5 cm 150 μm fused silica Kasil1 (PQ Corporation) frit trap loaded with 3 μm Reprosil-Pur C18 (Dr. Maisch) reverse-phase resin analyzed with a Thermo Easy nano-LC 1200. The PRTC mixture is used to assess quality of the column before and during analysis. Four of these quality control runs are analyzed prior to any sample analysis and then after every six to eight sample runs another quality control run is analyzed. Buffer A was 0.1% formic acid in water and buffer B was 0.1% formic acid in 80% acetonitrile. The 40-minute QC gradient consists of a 0 to 16% B in 5 minutes, 16 to 35% B in 20 minutes, 35 to 75% B in 1 minute, 75 to 100% B in 5 minutes, followed by a wash of 9 minutes and a 30 minute column equilibration. The 110-minute sample LC gradient consists of a 2 to 7% for 1 minutes, 7 to 14% B in 35 minutes, 14 to 40% B in 55 minutes, 40 to 60% B in 5 minutes, 60 to 98% B in 5 minutes, followed by a 9 minute wash and a 30 minute column equilibration. Peptides were eluted from the column with a 50°C heated source (CorSolutions) and electrosprayed into a Thermo Orbitrap Fusion Lumos Mass Spectrometer with the application of a distal 3 kV spray voltage. For the quality control analysis, a cycle of one 120,000 resolution full-scan mass spectrum (350-2000 m/z) followed by a data-independent MS/MS spectra on the loop count of 76 data-independent MS/MS spectra using an inclusion list at 15,000 resolution, AGC target of 4e5, 20 sec maximum injection time, 33% normalized collision energy with a 8 m/z isolation window. For the sample digest, first a chromatogram library of 6 independent injections is analyzed from a pool of all samples within a batch. For each injection a cycle of one 120,000 resolution full-scan mass spectrum with a mass range of 100 m/z (400-500 m/z, 500-600 m/z…900-1000 m/z) followed by a data-independent MS/MS spectra on the loop count of 26 at 30,000 resolution, AGC target of 4e5, 60 sec maximum injection time, 33% normalized collision energy with a 4 m/z overlapping isolation window. The chromatogram library data is used to quantify proteins from individual sample runs. These individual runs consist of a cycle of one 120,000 resolution full-scan mass spectrum with a mass range of 350-2000 m/z, AGC target of 4e5, 100 ms maximum injection time followed by a data-independent MS/MS spectra on the loop count of 76 at 15,000 resolution, AGC target of 4e5, 20 sec maximum injection time, 33% normalized collision energy with an overlapping 8 m/z isolation window. Application of the mass spectrometer and LC solvent gradients are controlled by the ThermoFisher XCalibur data system. Thermo RAW files were converted to mzML format using Proteowizard (version 3.0.20064) using vendor peak picking and demultiplexing. Chromatogram spectral libraries were created using default settings (10 ppm tolerances, trypsin digestion, HCD b- and y-ions) of EncyclopeDIA (version 0.9.5) using a Prosit predicted spectra library based on Uniprot human canonical FASTA background (April 2019). Prosit library settings were 1 missed cleavage, 33% NCE, charge states of 2 and 3, m/z range of 396.4 to 1002.7, and a default charge state of 3. Quantitative spectral libraries were created by mapping spectral to the chromatogram spectral library using EncyclopeDIA requiring a minimum of 3 quantitative ions and filtering peptides at a 1% FDR using Percolator 3.01. The quantitative spectral library was imported into Skyline (daily version 20.1.9.234) with the human uniprot FASTA as the background proteome to map peptides to proteins. In Skyline, data was TIC normalized and unique peptides were summed to protein TAF (total area fragment) quantities. If a peptide mapped to more than one protein, Skyline selected the first protein on the list. A csv file of unique protein TAFs for each replicate was exported. Unique, TIC normalizes TAFs value csv file was sent to Lu Wang (Shock Center statistician) for statistical processing. Refer to zip file “Mammalian_cell_proteomics_analysis_Solesio_pid2054” for detailed information.

There are 2 batches for this dataset. Batch 1 is MS01-MS20, MSREF01, MSPR01 and batch 2 is MS21-MS40, MSREF02, MSPR02. There are 40 neuroblastoma mammalian cell samples (MS01-MS40), 1 reference for each batch of a pooled neuroblastoma mammalian cell sample set from a pilot assay (MSREF01 and MSREF02) and another reference for each batch that is a pool of the 20 samples in this study from batch 1 (MSPR01 and MSPR02). For the 40 samples there are 5 biological replicates from Day 1 and Day 2 for each condition. The samples are composed of a total of 4 total conditions - 2 genotype groups (Control vector-only AKA WT SH-SY5Y and MitoPPX SH-SY5Y) and 2 cellular states (control AKA non-senescent, 7 passages and senescent, 15 passages). Note – there are different number of cells for the different cellular states.