Maxis hd qtof

The reconstituted peptides were used for shotgun proteomics experiments for the identification of the endogenous peptides. The peptides were separated using micro-LC (Dionex, Thermo UltiMate 3000 HPLC System, USA) through analytical column (Supelco, Ascentis Express C18, 25 cm $\times$ 4.6 mm, 2.7 µm) coupled with ESI source (BrukerDaltonics, Germany) spray in Maxis-HD qTOF (Bruker, Germany) mass spectrometer. The acquisition parameters were adapted from our previous reports with slight modifications^{69 (link), 70 (link)}. The elution was performed with a flow rate of 150 µL/min a continuous gradient of 5–75% acetonitrile over 135 min. In the solvent system; Solvent A was 100% water with 0.1% formic acid, and solvent B was 100% acetonitrile with 0.1% formic acid. Data were acquired in the data-dependent mode in mass spectrometer operated in automatically switching between MS and MS/MS acquisition. The precursor ion MS spectra scan range of 200–2200 (m/z) was used in the Q-TOF with resolution R = 75,000. The six most abundant precursor ions were searched for detection of different masses during acquisition and selected for fragmentation using collision-induced dissociation (CID) with a fixed cycle time of 3 s along with 2 min of release for exclusion filter (otof processing software, BrukerDaltonics).

The lyophilized peptide fractions were reconstituted in 0.1% formic acid in LC-MS-grade water and subjected to nano-LC (Nano-Advance; Bruker, Germany) followed by identification in captive ion source (Bruker Captive Spray tip) spray-in Maxis-HD qTOF (Bruker) mass spectrometer (MS) with high mass accuracy and sensitivity. The peptides were enriched in nano-trap column (Acclaim Pep Map, particle size 5 μm, pore size 100 Å; Thermo Scientific) and eluted on to nano-analytical column (Kaya Tech HIQ SIL C₁₈HS/3, 0.1 × 150 mm, 3 μm particle size, and 200 Å pore size). The peptide elution was carried out using a linear gradient of 5–45% acetonitrile at 400 nl/min flow rate in a total run time of 135 keeping the solvent system as follows: solvent A, 100% water in 0.1% formic acid; and solvent B, 100% acetonitrile in 0.1% formic acid. Positive ions were generated by electrospray, and the q-TOF was operated in data-dependent acquisition mode to automatically switch between MS and MS/MS acquisition. Precursor ion TOF MS survey scan was acquired with a range of 300–1,800 m/z with resolution R = 75,000. Q1 sequentially selects six most intense precursor ions for fragmentation using collision-induced dissociation for MS-MS analysis with a fixed cycle time of 3 s along with 2 min of release for exclusion filter (Data acquisition otof software, version 24.8; Bruker Daltonics).

The reconstituted peptides were used for shotgun proteomics experiments for the identification of endogenous peptides. The peptides were separated using micro-LC (Thermo UltiMate 3000 HPLC System; Dionex, Sunnyvale, CA, USA) through an analytical column (Supelco Sigma-Aldrich, St. Louis, MO, USA; Ascentis Express C18, 25 cm×4.6 mm, 2.7 μm) coupled with ESI source (Bruker Daltonics, Germany) spray in Maxis-HD qTOF (Bruker, Germany) mass spectrometer. The elution was performed with a flow rate of 150 μL/min and a continuous gradient of 5% to 75% ACN over 135 min. In the solvent system, solvent A was 100% water with 0.1% formic acid, and solvent B was 100% ACN with 0.1% formic acid. Data were acquired in the data-dependent mode in a mass spectrometer that operated automatically switching between MS and MS/MS acquisition. The precursor ion MS spectra scan range of 200 to 2,200 (m/z) was used in the Q-TOF with resolution R = 75,000. The six most abundant precursor ions were searched for detection of different masses during acquisition and selected for fragmentation using collision-induced dissociation with a fixed cycle time of 3 s along with 2 min of release for exclusion filter (Q-TOF processing software; Bruker Daltonics, Germany).