Q exactive plus orbitrap

A custom-built nano-DESI MSI source was assembled as previously described [11 (link)], and a capillary holder was used to fix the primary and secondary capillaries at an angle [18 (link)]. The nano-DESI solvent consisted of a 9:1 solution of methanol:water (v/v), with an addition of 3 μmol/l lysophosphatidylcholine 19:0, and was supplied at a rate of 0.5 μl/min. Imaging was performed by moving the sample in lines under the nano-DESI probe at a speed of 60 μm/s in the x-direction and a step size of 200 μm between the lines in the y-direction. The average acquisition time of 0.7 s per spectrum resulted in pixel sizes of ~ 42 × 200 μm. Three tissue sections from one kidney each of 3 control and 3 STZ-treated rats were analysed by nano-DESI MSI in a random order.
All mass spectrometric analyses were performed in an untargeted fashion on a Q-Exactive™ Plus Orbitrap™ (Thermo Fisher Scientific, Bremen, Germany). MSI data acquisition was performed in positive mode with a scan window of m/z 100–1000, using a mass resolution of 140,000 (m/Δm at m/z 200). The instrument was externally calibrated, the spray voltage was set to 3 kV, and the heated capillary temperature was set to 300 °C.

Typically, 10 µL of sample was injected into the LC–MS/MS system consisting of a Dionex UltiMate 3000 Rapid Separation LC (RSLC) system with an Orbitrap Fusion or Q Exactive Plus Orbitrap (Thermo Scientific). Peptides were trapped using an Acclaim PepMap100 C18 Trap column (Thermo Scientific) at a flow rate of 15 μL/min using 0.1% formic acid/2% acetonitrile in H₂O (solvent A). Peptides were eluted from the trap column to an in-house packed column (75 µm inner diameter (ID) × 25 cm; 1.9 µm C18 media, Dr Maisch) with a pulled tip emitter. Peptides were eluted from the column using a linear 100-min gradient from 5% to 40% solvent B (0.1% formic acid/80% acetonitrile). Data was collected in positive mode and full spectra acquired from m/z 350 to 2000, with a resolution of 70,000. For MS/MS, the top 15 most intense precursor ions were fragmented and ionised, and MS/MS spectra with m/z 350–1750 and resolution 17,500 recorded.

To a previous report 20 (link), the aliquots extracts were dissolved in methanol at 10 μg/ml and conducted on a Q-Exactive Plus orbitrap high-resolution mass spectrometer equipped with an Ultimate 3000 ultra-high performance liquid chromatographic (UHPLC) system (ThermoScientific, San Jose, CA, USA) and a Waters ACQUITY UPLC C18 column (2.1 × 100 mm, 1.7 um). The adopted gradient was set as follows: from 5% buffer B at 1 minute to 30% buffer B at 15 minutes, subsequently to 80% buffer B at 16 minutes, and maintained at 80% buffer B for 5 minutes, where buffer A was 0.2% formic acid/H2O, and buffer B was acetonitrile. The flow rate of 0.4 mL/min. The parallel reaction monitoring (PRM) mode of Q-Exactive Plus was utilized to characterize chemical components and quantified caffeine, chlorogenic acid (CGA), quinic acid, and mangiferin. The survey scan was conducted in the m/z 150-2000, with an electrospray voltage of 4.0 kV and 3.5 kV for positive and negative ionization modes, respectively. The standard pure compounds, caffeine, chlorogenic acid, and quinic acid, were purchased from Sigma-Aldrich, USA. The mangiferin was acquired from ChromaDex, California, USA.

Samples were run on a Q-Exactive Plus Orbitrap (Thermo Scientific, Waltham, MA, USA) equipped with EASY-nLC 1000 UHPLC. Separation of peptides was performed on an Acclaim PepMap RSLC 150 mm C18 column, 50 µm of internal diameter (2 µm bead diameter, 100 A) with a flow rate of 200 nL/min and in a gradient of 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitril (buffer B). Content of buffer B was increased from 2% to 30% during 240 min and from 40% to 100% in 5 min. The sample load was 5 µL. The mass spectrometer was operated in the data dependent mode with automatic switching between full scan MS and MS/MS acquisition. Survey full scan MS spectra (m/z 300−1800) were acquired in the Orbitrap with a resolution of 70,000 (m/z 200) after accumulation of ions to a target value of 3 × 10⁶, based on predictive AGC from the previous full scan. Dynamic exclusion was set to 20 s. The 12 most intense multiply charged ions (z ≥ 2) were sequentially isolated and fragmented in the octopole collision cell, by higher-energy collisional dissociation (HCD) with a maximum injection time of 120 ms. A range from 50 to M + 50 Da was covered for MS2 (resolution of 17,500). A 2.5 Da isolation width was chosen.

Dried peptides were resuspended in 30 µL of 0.1% FA and analyzed using a Q-Exactive Plus Orbitrap hybrid mass spectrometer (Thermo Scientific) equipped with an Ultimate 3000 nano-high-pressure liquid chromatography (nano-HPLC) system (Dionex), HTC-PAL autosampler (CTC Analytics), and a nanoelectrospray ion source. Five microliters of each sample were injected into a Zorbax 300SB C18 capillary column (0.3 × 150 mm; Agilent Technologies) and heated at 40 °C. A 1-h HPLC gradient was employed (1% B to 32% B in 45 min, 32% B to 45% B in 15 min, with final wash at 75% B for 5 min and reequilibration at 1% B for 10 min.) using 0.1% FA in distilled water as solvent A, and 0.1% FA in ACN as solvent B. A flow rate of 3.5 μL/min was used for peptide separation. Temperature of the heated capillary was 300 °C, and a 1.9-kV spray voltage was applied to all samples. The mass spectrometer settings were as follow: full MS scan range 350 to 1,500 m/z with a mass resolution of 70,000, 30-μs scan time, and automatic gain control set to 1.0E6 ions, and fragmentation MS2 of the 20 most intense ions.