Rslcnano system

TRPM6 and TRPM7 proteins were analyzed on a Nano-LC-MS/MS system that included a Nano-liquid chromatograph (Dionex Ultimate 3000, RSLCnano System, Thermo Fisher Scientific Inc.) and a CaptiveSpray source/Quadrupole ion trap mass spectrometer (Model Q-ToF Com-pact II, Bruker, Hamburg, Germany). Peptides were enriched by the Nano trap column and separated on a PepMap100 C18 LC column. Peptides were eluted at a flow rate of 300 nL/min at 60°C under a linear gradient of 2%–95% Solvent B over a 90 min run of mobile phase A. Mobile phase A consisted of water/FA (99.9:0.1, v/v) while solvent B was composed of acetonitrile/water/FA (80:19.92:0.08, v/v). Mass spectral data from the 300 to 2,200 m/z range were collected in the positive ionization mode with acquisition rate set at 6 Hz. Auto MSN CID fragmentation experiments were performed at low (4 Hz) and high (16 Hz) mass spectral rates for the top 2 most intense precursor ions using 3 sec dynamic exclusion. Peptide sequences were matched on the UniProtKB database (https://www.uniprot.org/help/uniprotkb) using the MASCOT (v 2.3) searching engine (Matrix Science Ltd., London, UK). Exponentially modified protein abundance index (emPAI) was used to determine protein abundance in each LC-MS/MS experimental sample (29 (link)), while phosphorylation and oxidation of TRPM6 and TRPM7 were determined by MS/MS fragmentation analysis.

The pellets of purified GC-VLNs were lysed in NuPAGE™ LDS sample buffer (ThermoFisher Scientific, Waltham, MA, USA) and the lysate was run in a Bolt™ 12% Bis-Tris Plus gel for 10 min, followed by in-gel digestion with trypsin. The obtained peptides were subjected to LC-MS/MS analysis using a RSLCnano system (ThermoFisher Scientific) coupled to a Q-Exactive HF mass spectrometer (ThermoFisher Scientific). The resulting proteomics data were analyzed with Mascot v 2.6.1 (Matrix Science, Boston, MA, USA) using the common contaminants database cRAP (123 entries, www.theGPM.org) and the Uniprot entries for viridiplantae (retrieved on 04/30/2019, 7108828 entries). The identified peptides and proteins were validated using Scaffold v4.8.9 (Proteome Software Inc., Portland, OR). For peptides, if they reached greater than 80.0% probability by the Peptide Prophet algorithm 75 (link) with Scaffold delta-mass correction, their identifications were accepted. For proteins, if they reached 99.0% probability by the ProteinProphet algorithm 76 (link) and contained at least 2 identified peptides, their identifications were established.

LC-MS/MS analyses were carried out using an RSLCnano System (Dionex, UK) and an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Samples were directly loaded and eluted at a flow rate of 300 nL/min onto a reverse-phase column (75 μm i.d., 450 mm) packed with ProntoSIL C18AQ 3 μm media (Bischoff, Germany) that was prepared in-house. The peptides were eluted with buffer A (0.1% formic acid in H₂O) and buffer B (0.1% formic acid in CH₃CN) in the following gradient segments of buffer B: 17 mins, 0–2%; 60 mins, 2–25%; 2 mins, 25–44%; 2 mins, 44–76%; 3 mins, 76%; and 2 mins, 76–2%. The eluted peptides from the column were sprayed into a nanospray ion source on an Orbitrap Fusion Tribrid mass spectrometer set to record single microscan FTMS scan events at a resolution of 30000 over the m/z range 300–1500 Da in positive ion mode, with charge states of 2–7 included. The top 15 data-dependent CID MS/MS were triggered from the FTMS scan and introduced into the Orbitrap- Fusion ion trap. The collision energy was set to 35% and activation Q to 0.25 with the scan range and ion trap scan rate both set to normal. The automatic gain control (AGC) target values were set to optimal conditions using 500,000 for MS1 and 5,000 for MS251 (link). Dynamic exclusion was allowed once for a 90-second duration.

Histone peptides were resuspended in 0.1% trifluoroacetic acid prior to mass spectrometric analysis on a ThermoFisher Scientific TSQ Quantum QqQ MS (San Jose CA). Using a Dionex RSLCnano system (Sunnyvale CA), peptides were loaded onto a trapping column (3cm x 150μm) and separated on a PicoChip analytical column (10cm x 75μm), both packed with ProntoSIL C18-AQ, 3μm, 200Å pore size (New Objective, Woburn MA). Elution occurred over a chromatography gradient of buffer B from 0 to 35% at a flow rate of 0.30 μL/min over 45 minutes. Solvent A: 0.1% formic acid in water, and B: 0.1% formic acid in 95% acetonitrile. The peptides were then introduced into the QqQ MS by electrospray from an integrated emitter with 10 μm tip (New Objective) after elution from the analytical column. Targeted analysis of unmodified and various modified histone peptides was performed with the following settings: collision gas pressure of 1.5 mTorr; Q1 peak width of 0.7 (FWHM); cycle time of 3 s; skimmer offset of 10 V; electrospray voltage of 2.5 kV. Histone peptides (modified and unmodified) were selected for monitoring based on previous publications [20 (link),21 (link)]. A list of all peptides monitored in the assay can be found in S1 Table.