Q exactive hf orbitrap ms

The fractionated samples were processed by acidic reverse-phase LC–MS/MS coupled with a Q Exactive HF Orbitrap MS (Thermo Fisher Scientific), using a self-packed column (75 μm × 150 mm, 1.9 μm C18 resin from Dr Maisch GmbH, at 65 °C). Peptides were eluted with 70 min gradient (buffer A: 0.2% formic acid, 5% dimethyl sulfoxide; buffer B: buffer A plus 65% acetonitrile). The setting of MS involved positive ion mode and data-dependent acquisition (top20: one MS1 scan followed by 20 MS/MS scans). MS1 scans were executed at a 60,000 resolution, 460 to 1600 m/z scan range, a maximum ion time of 50 ms, and 1 × 10⁶ automatic gain control. MS2 scans were acquired at a 60,000 resolution, a fixed initial mass of 120 m/z, a maximum ion time of 120 ms, and 1 × 10⁵ automatic gain control. The fragmentation settings included 1.0 m/z isolation window with 0.2 m/z offset, normalized collision energy of 32, and 15 s of dynamic exclusion.

The pooled TMT labeled peptides were fractionated by an offline basic pH RPLC with a XBridge C18 column (3.5 μm particle size, 4.6 mm × 25 cm, Waters; buffer A: 10 mM ammonium formate, pH 8.0; buffer B: 90% AcN, 10 mM ammonium formate, pH 8.0). The peptides were eluted in a 160 min gradient of 15–50% buffer B, and 160 fractions were collected every min and concatenated to 40 fractions. Each fraction was analyzed on a self-packed column (75 μm × 15 cm with 1.9 μm C18 resin) coupled with a Q Exactive HF Orbitrap MS (Thermo Fisher Scientific). The peptides were eluted at 0.25 μL/min flow rate with a 60 min gradient of 18–45% buffer B (buffer A: 0.2% formic acid, 5% DMSO; buffer B: buffer A plus 65% AcN). The mass spectrometer was operated in data-dependent mode with a MS1 scan in Orbitrap mass analyzer [450–1600 m/z; 60,000 resolution; 1 × 10⁶ automatic gain control (AGC) target; 50 ms maximum ion time] and 20 data-dependent MS2 scans (60,000 resolution, 1 × 10⁵ AGC target, 110 ms maximum ion time, 32% HCD normalized collision energy (NCE), 1.0 m/z isolation window, 0.2 m/z isolation offset, and 10 s dynamic exclusion).

Samples were vacuum desiccated for 30 min and then washed in fresh solutions of 70% ethanol for 30 s, 100% ethanol for 30 s, Carnoy’s solution (6:3:1 v/v ethanol/chloroform/glacial acetic acid) for 2 min, 100% ethanol for 30 s, water with 0.2% TFA for 15 s, and 100% ethanol for 30 s. Samples were then dried by a stream of nitrogen gas prior to MALDI matrix application. HTX Technologies M5 Sprayer was used to deposit sonicated supernatant of 15 mg/ml 2,5-DHA (2,5-dihydroxyacetophenone) in 90% acetonitrile with 0.2% TFA. The flow rate of the matrix was 150 μl/min with a nozzle temperature of 30.0 °C, with a velocity set to 1300 mm/min with 10 PSI of nitrogen gas. The matrix was then recrystallized with 5% acetic acid solution in water at 38.5 °C and dried for 3.5 min and then immediately analyzed using an elevated pressure MALDI source (Spectroglyph LLC) coupled to a Thermo Scientific Q Exactive HF Orbitrap MS upgraded with ultra-high mass range boards (28 (link)). Spectra were acquired over the m/z range of 3500 to 20,000 in positive polarity mode with a resolving power of 240k at m/z 200 (512 ms transient) and 250 laser shots per pixel. Scans in the.RAW file were summed as a single spectrum for proteoform assignment by accurate mass.

Cells were cultured in 6-well plates to ~85% confluence and washed with 2 mL ice cold 1X Phosphate-Buffered Saline (PBS). The cells were then harvested in 300 µL freezing 80% acetonitrile (v/v) into 1.5 mL tubes and lysed by Bullet Blender (Next Advance) at 4 °C followed by centrifugation at 21,000 × g for 5 min at 4 °C. The supernatant was dried by speedvac and reconstituted in 7.5 µL of 66% acetonitrile and 2 µL was separated by a ZIC‐HILIC column (150 × 2.1 mm, EMD Millipore) coupled with a Q Exactive HF Orbitrap MS (Thermo Fisher) in negative detection mode. Metabolites were eluted within a 45 min gradient (buffer A: 10 mM ammonium acetate in 90% acetonitrile, pH = 8; buffer B: 10 mM ammonium acetate in 100% H2O, pH = 8). The MS was operated by a full scan method followed by targeted selected ion monitoring and data-dependent MS/MS (tSIM/dd-MS2). MS settings included full scan (120,000 resolution, 350–550 m/z, 3 × 106 AGC and 50 ms maximal ion time), tSIM scan (120,000 resolution, 1 × 105 AGC, 4 m/z isolation window and 50 ms maximal ion time) and data-dependent MS2 scan (30,000 resolution, 2 × 105 AGC, ~50 ms maximal ion time, HCD, Stepped NCE (50, 100, 150), and 10 s dynamic exclusion). Data were quantified using Xcalibur software (Thermo Fisher Scientific) and normalized by cell numbers. Ribonucleotide and deoxyribonucleotides were validated by authentic standards.