Rapiflex maldi tof tof

Tissue sections of sprayed matrix were placed in a Bruker Rapiflex MALDI-TOF/TOF type mass spectrometer (Bruker, Karlsruhe, Germany) equipped with a smartbeamTM 3D laser for mass spectrometry imaging analysis. The wavelength was 355 nm at a sampling rate of 2.5 GS/s. MALDI mass spectrometry imaging was performed in a positive ion detection mode with a mass-to-charge ratio (m/z) ranging from 100 to 1500 and a spatial resolution of 100 μm for scanning samples.

Matrix-assisted laser desorption ionization with time-of-flight analysis was performed on a rapifleX MALDI-ToF/ToF from Bruker. Atmospheric pressure chemical ionization MS was recorded with atmospheric pressure solids analysis probe using an Advion expression compact mass spectrometer.

We added 0.2 µL of 20 mg/mL 2,5-dihydroxybenzoic acid (DHB, Sigma-Aldrich) in 70% LC-MS grade water with 0.1% trifluoroacetic acid (Acros Organics) and 30% acetonitrile (VWR) to the individual spots on a ground steel MALDI target plate. Mass spectra were acquired from 140 m/z to 460 m/z using a Bruker rapifleX MALDI-TOF/TOF in positive reflector mode (Bruker Daltonik GmbH). Each mass spectrum consisted of 1000 shots performed in a 10-µm square region using a 10-kHz laser repetition frequency. Mass spectra were baseline subtracted and calibrated to DHB matrix ions with the mMass software tool^{51 (link)}. For comparisons between donor and acceptor droplets, intensity values were normalized to the internal standard peak of HEPES (239.1 m/z). The detected and theoretical monoisotopic peaks are compared in Table S2 for analytes l-histidine, l-arginine, HEPES, and riboflavin. All detected analyte peaks had an m/z error of <0.025.

CrRNAs were extracted from purified Cas10-Csm^Csm3D32A with 1:1 (v/v) ratio of phenol–chloroform–isoamyl alcohol (25:24:1) twice, followed by one extraction with 1 vol. chloroform. For PAGE analysis, crRNAs were ³²P-labelled by incubation with γ-³²P-ATP (3000 Ci/mmol) and T4 Polynucleotide kinase at 37°C for 1 h. Extracted crRNAs were mixed 1:1 with formamide dye (5 mM EDTA pH 8.0, 95% formamide v/v) and heated at 70°C for 2 min before loading to a 12% acrylamide, 8 M urea gel. Invitrogen Decade markers were added to the gel to infer crRNA sizes. Electrophoresis was carried out at 50 W for approximately 90 min. Phosphorimaging was performed with storage phosphor screens and a Typhoon FLA 7000 both from GE Healthcare.
For mass spectrometry analysis, crRNAs were desalted with C18 ziptips according to the manufacturer’s instructions. One microlitre of matrix (9 parts of 50 mg/mL 3-hydroxypicolinic acid in 50% acetonitrile/water and 1 part 50 mg/mL ammonium citrate in water) was spotted on a matrix-assisted laser desorption ionization (MALDI) target and was dried by a stream of nitrogen gas. One microlitre of desalted crRNAs was then layered on the dried matrix and the mixture was subjected to MALDI mass spectrometry with a Bruker RapifleX MALDI TOF/TOF.