Ftmscontrol 2

MSI experiments were carried out using a Bruker SolariX 7T XR hybrid ESI–MALDI–FT–ICR–MS equipped with a SmartBeam II UV laser. MSI analysis in positive ionization mode was carried out using optimized instrumental settings for the mass range 100–1500 m/z in broadband mode with a Time Domain for Acquisition of 2 M providing an estimated resolving power of 130,000 at 400 m/z. The laser was set to 50% power using the minimum spot size (an ovaloid shape with approximately 10 × 15 µm dimensions), 750 shots per sample spot at a frequency of 2 kHz were collected, MALDI smart walk with a 25 µm grid width, 10% grid increment and a smart walk pattern grid offset of one was enabled to enhance sampling of the MALDI spot providing an ablation spot of less than 30 × 30 µm. Mass spectra were acquired using Bruker Daltonics ftmsControl 2.1.0.

MALDI-MSI analysis was performed on a Bruker SolariX (7T XR hybrid ESI–MALDI–FT–ICR–MS) with a mass resolving power of 200,000 and equipped with a SmartBeam II UV laser. Before data collection, the instrument was calibrated with a red phosphorus standard to ensure that its mass error was less than 1.5 ppm. Two technical replicates were imaged per sample and two samples were imaged per MSI run. The area-of-interest for imaging was defined using flexImaging 4.1 (Bruker Daltonics) and data acquisition was controlled via Bruker Daltonics ftmsControl 2.1.0. Spectra were collected at a spatial resolution of 50 μm and a range of 150–2,000 m/z under positive ion mode. Laser diameter and power were set to 45 μm and 38% (E. diaphana) or 52% (G. fascicularis), and a total of 250 (E. diaphana) or 500 (G. fascicularis) laser shots were applied at each 50 μm pixel at a frequency of 2 kHz.

Bands corresponding to crosslinked protein were excised from the SDS-PAGE gels and distained. Cysteines were reduced with 50 mM DTT for 45 min at 60°C and free cysteines were alkylated with 100 mM iodoacetamide for 30 min at room temperature in the dark. Trypsin digestion proceeded overnight at 37°C with an enzyme/protein ratio of 1:20 (w/w). Peptides extracted from the gel were loaded on a trap column (ZORBAX 300SB-C18, 5 μm, 5 × 0.3 mm, Agilent, Santa Clara, CA) and desalted for 5 min at flow rate 20 μL/min. Peptides were then separated by reversed phase C18 column (ZORBAX SB C18 RR 3.5 μ 150 × 0.3 mm, Agilent, Santa Clara, CA) at a flow rate 10 μL/min using capillary HPLC system (Agilent Technologies) under the following gradient conditions: 1–10% B in 1 min, 10–45% B in 19 min, 45–95% B in 5 min, where solvent A was 0.1% formic acid, 2.0% acetonitrile in water and solvent B was 0.1% formic acid in 98% acetonitrile. The column was heated at 50°C and connected directly to an 15T solariX FT-ICR mass spectrometer (Bruker Daltonics) using an electrospray ion source. The instrument was on line calibrated resulting in mass accuracy below 2 ppm. Data acquisition and data processing were performed by ftmsControl 2.1.0 and DataAnalysis 4.2 (Bruker Daltonics).