Rapiflex maldi tof mass spectrometer

LCTEM chips, with their SiN_x membranes facing upwards were adhered to the conductive face of an ITO-coated glass slide with 70–100 ohms resistivity (Bruker Daltonics), using ~0.5 µL nail polish and allowed to dry. To equalize the height difference from SiN_x chips on the slide (~0.25 mm), four pieces of Scotch tape were applied to both short edges of the slide on the same side. As a control, unimaged PDEGMA in deionized water was drop casted onto a clean liquid-cell chip. All chips were coated with trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) matrix in (20 mg mL^− 1 in acetonitrile).
Slides were mounted into an MTP Slide Adapter II and loaded onto a Bruker Rapiflex MALDI-ToF mass spectrometer for analysis using the flexControl software (Bruker Daltonics). Samples were analyzed by MALDI-MS under either reflector positive mode (1400–10000 Da) using a 355 nm smartbeam 3D laser with a 50 µm focus diameter and 200 Hz frequency, a constant laser power of 75%, and a sum of 500 shots per spectrum. Spectra were collected using an accelerating voltage of 20 kV and detector gain of 792 V. Region of interest (ROI) mapping was performed at a raster width of 50 µm, and image analysis was performed in flexImaging software (Bruker Daltonics).

The high-performance liquid chromatography (HPLC) was used to determine free amino acid analysis of CV. Weigh 1 g of powder, add 10 times the amount of distilled water, and heat to solidify, and the solution is filtered to obtain an aqueous layer. The residue obtained by concentrating the aqueous layer to dryness under reduced pressure was dissolved in 0.2 N sodium citrate buffer (pH 2.2) and used as a sample for HPLC. Agilent LC system (Agilent Technologies, Santa Clara, CA, USA) and Capcellpak UG120 with properties of (250 mm × 4.6 mm × 5 μm) was used. The mobile phases were: A, 40 mM NaH2PO4 (pH 7.8) and B ACN:MeOH:DW (45:45:10 v/v) and the elution gradient was: 0–31 min, 95% A; 34 min, 44% A; 38 min, 0% A. The flow rate was 1.5 mL/min, and the absorbance of detection was 262 and 338 nm. Column temperature was set to 40 °C and injection volume was 10 μL.
Peptide molecular weights of CVs were measured in linear mode using a RapifleX MALDI-TOF mass spectrometer (Bruker, Billerica, MA, USA). Sample preparation and peptide analysis were performed by modifying method Lu et al. [17 (link)].

For MALDI‐TOF sample preparation, a mixture containing a 1:1 ratio of 2% (v/v) TFA and 2,5 dihydroxyacetophenone (DHAP) matrix solution (7.6 mg of 2,5 DHAP in 375 μl 100% (v/v) ethanol and 12 μl of 12 mg/ml diammonium hydrogen citrate) was added to the sample in a 1:1 ratio. One microlitre of the solutions were spotted in duplicate onto an MTP AnchorChip 1,536 Target (Bruker Daltronics). Samples were air dried at room temperature prior to analysis. All spectra were acquired using a Rapiflex MALDI‐TOF mass spectrometer (Bruker Daltronics) equipped with Compass 1.3 control. Recording took place in automatic mode (AutoXecute, Bruker Daltronics), allowing 6–9 s per target spot as described previously (De Cesare et al, 2020 (link)). Spectra were visualised using FlexControl software and processed by FlexAnalysis software (version 4.0).