Ultraflex 3 maldi tof spectrometer

C₆₀ (99.5%) and C₇₀ (95%) were purchased from SES Research and MER corporation respectively. CH₂Cl₂ was freshly distilled over CaH₂ before use. All other reagents and solvents were purchased from Aldrich and used without further purification. Compounds 1–3 and 7–10 were synthesised according to previously reported procedure [20 (link)]. Infra-red spectra were measured as KBr discs using a Nicolet Avatar 380 FTIR spectrometer over the range 400–4000 cm⁻¹. ¹H and ¹³C NMR spectra were obtained using Bruker DPX 300, Bruker DPX 400, Bruker AV(III) 400 or Bruker AV(III) 500 spectrometers. Mass spectrometry was carried out using a Bruker microTOF spectrometer and a Bruker ultraFlexIII MALDI–TOF spectrometer using trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) as supporting matrix. UV–vis spectra were measured using a Lambda 25 Perkin Elmer Spectrometer. EPR spectra were obtained on a Bruker EMX EPR spectrometer.

Sample preparation and analysis by MALDI-TOF MS protein profiling followed a protocol optimized for sand flies [12 (link)]. In total, 20 specimens (10 collected in 2016, 10 collected in 2017) were analyzed. Thoraxes of dissected specimens were homogenized by a manual BioVortexer homogenizer (BioSpec, Bartlesville, USA) with sterile disposable pestles in 10 μl of 25% formic acid and briefly centrifuged at 10,000g for 15 s. Two microliters of the homogenate was mixed with 2 µl of freshly prepared MALDI matrix, which was an aqueous 60% acetonitrile/0.3% TFA solution of sinapinic acid (30 mg/ml, Bruker Daltonics, Bremen, Germany). One microliter of the mixture was then spotted on a steel MALDI plate in duplicate. Protein mass spectra were measured in a mass range of 4–25 kDa on an Ultraflex III MALDI-TOF spectrometer (Bruker Daltonics) as a sum of 2000 laser shots (20 × 100 shots from different positions of the sample spot) and analyzed by FlexAnalysis 3.4 software. For species identification and cluster analysis, the protein profiles were processed using MALDI Biotyper 3.1 and compared with reference spectra of an in-house database constructed using protein spectra of 25 different sand fly species. For MSP dendrogram creation, an individual main spectrum (MSP) was generated from each analyzed spectrum.

Two μl of the ethanol or the water protein extract were mixed with 2 μl of a MALDI matrix in a tube. One μl of the resulting mixture was deposited on the MALDI target and allowed to air-dry. The MALDI matrix was prepared daily as an aqueous 60% acetonitile/0.3% TFA solution of sinapinic acid (30 mg/ml; Sigma). Positive-ion mass spectra were measured in linear mode on an Ultraflex III MALDI-TOF spectrometer (Bruker Daltonics, Bremen, Germany) within a mass range of 2–25 kDa and calibrated externally using the Bruker Protein Calibration Standard I. Each acquired spectrum corresponded to an accumulation of 1000 laser shots (5×200 laser shots from different positions of the target spot). The spectra were exported to the MALDI Biotyper 3.1 software for data processing (normalization, smoothing, baseline subtraction, peak picking) and evaluation by cluster analysis. Only a maximum of 100 peaks with signal-to-noise ratio of >3 and relative intensity of at least 0.1% of the most intense peak from the spectra were considered for choosing peaks. For MSP dendrogram creation, an individual main spectrum was generated from each of the acquired spectra.