Thermo q exactive hf mass spectrometer

Each sample was analyzed four times using HILIC and RPLC separation in both positive and negative ionization modes. Data were acquired on a Thermo Q Exactive HF mass spectrometer for HILIC and a Thermo Q Exactive mass spectrometer (Thermo Fisher Scientific) for RPLC. Both mass spectrometers were equipped with a HESI-II probe and operated in full MS scan mode. For HILIC experiments, a ZIC-HILIC column (2.1 × 100 mm, 3.5 μm, 200 Å; EMD Millipore) was used with mobile phase solvents (A) 10 mM ammonium acetate in 50:50 acetonitrile:water and (B) 10 mM ammonium acetate in 95:5 acetonitrile:water. For RPLC experiments, a Zorbax-SB-aq column (2.1 × 50 mm, 1.7 μm, 100 Å; Agilent) used with mobile phase solvents (A) 0.06% acetic acid in water and (B) 0.06% acetic acid in methanol. Before running the sequences, LC-MS systems were equilibrated by injecting 12 and 6 pooled quality control samples (QCs) for HILIC and RPLC, respectively. MS/MS data were acquired on pooled QCs consisting of an equimolar mixture of all the samples in the study at normalized collision energies (NCE) of 25 and 35 for HILIC and 25 and 50 for RPLC. Multiple quality control measures were performed to ensure data quality. All samples were randomized prior to protein extraction and data acquisition. Further, mass accuracy, retention time and peak shape of internal standards were reviewed in each sample.

Samples (dissolved in H₂O with 0.1% FA) (n=7) were analyzed on both a Thermo MALDI-LTQ-Orbitrap XL and a Thermo Q Exactive HF mass spectrometer (Thermo Scientific, Bremen, Germany) for MALDI-MS and LC-ESI-MS/MS, respectively. For analysis on the MALDI-LTQ-Orbitrap XL (equipped with a 337.1 nm, 60 Hz nitrogen laser), samples were spotted with DHB (150 mg/mL; 50:50 MeOH:H₂O and 0.1% FA) at a 1:1 (v:v) ratio in triplicate. For analysis on the Q Exactive HF, the instrument was coupled to a nano-ESI source connected to an online Dionex UltiMate 3000 nanoLC and each biological sample was injected 3 times for 3 technical replicates. All major instrument parameters for MALDI- and LC-ESI-MS/MS can be found in Tables S8 and S9, respectively.

Plasma samples were analyzed using HILIC and RPLC separation in both positive and negative ionization modes. HILIC data were acquired on a Thermo Q Exactive HF mass spectrometer while RPLC data were acquired using a Thermo Q Exactive mass spectrometer (Thermo Fisher Scientific). Both mass spectrometers were equipped with a HESI-II probe and operated in full MS scan mode. HILIC experiments used a ZIC-HILIC column (2.1 × 100mm, 3.5 mm, 200 Å; EMD Millipore) that used mobile phase solvents (A) 10 mM ammonium acetate in 50:50 acetonitrile:water and (B) 10 mM ammonium acetate in 95:5 acetonitrile:water. RPLC experiments used a Zorbax-SB-aq column (2.1 × 50mm, 1.7mm, 100Å; Agilent) that used mobile phase solvents (A) 0.06% acetic acid in water and (B) 0.06% acetic acid in methanol. Before running any samples, LC-MS systems were calibrated, and the columns were equilibrated by injecting 12 and 6 pooled (QCs) for HILIC and RPLC, respectively. Pooled QCs were also used to acquire MS/MS data at normalized collision energies (NCE) of 25 and 35 for HILIC and 25 and 50 for RPLC.