Microflex lrf mass spectrometer

Intact mitochondrial enriched fractions from gastrocnemius muscle were deposited on the MALDI target with a “double layer” deposition method as follows: a 1 μL droplet of the mitochondrial enriched fractions suspension, at concentration of 0.4 mg/mL, was deposited on the MALDI target and dried under a cold air stream (first layer); the resultant solid deposition was then covered by a thin second layer (0.35 μL droplet) of the 9-AA matrix solution (20 mg/mL in 2-propanol-acetonitrile, 60:40, v/v). After solvent evaporation, the sample was analyzed. MS settings: MALDI-TOF mass spectra were acquired on a Microflex LRF mass spectrometer (Bruker Daltonics, Hamburg, Germany). The system utilizes a pulsed nitrogen laser, emitting at 337 nm; the extraction voltage was 20 kV, and gated matrix suppression was applied to prevent detector saturation. The laser fluence was kept about 10% above threshold to have a good signal-to-noise ratio. All spectra were acquired in the reflector mode using delayed pulsed extraction; spectra acquired in negative ion mode are shown in this study. Spectral mass resolutions and signal-to-noise ratios were determined by the software for the instrument, “Flex Analysis 3.3” (Bruker Daltonics).

Lipid A samples were dissolved in 90 μL of chloroform/methanol (1:1 v/v) and this solution was mixed with one volume of matrix (10 mg/mL 2.5-dihydroxybenzoic acid in 100 mM citric acid). Spectra were obtained in the negative reflection mode using a Microflex LRF mass spectrometer (Bruker Daltonics Inc., MA, United States). flexControl 3.0 (Bruker Daltonik GmbH, Germany) and mMass version 5.5.0 ¹ software packages were used for spectrometer control and spectra analysis, respectively.

MePCE (161-179) R171-me2s/C177S peptide, mixed with EDTA-free cOmplete protease inhibitor (Roche), αKG, Zn²⁺, and HEPES pH 6.5, was treated with recombinant wild-type JMJD6, inactive mutant JMJD6, or peptide alone and placed in 37°C for 2 hr. 1 μL of reaction sample is mixed with 1 μL of a-cyano-4-hydroxycinnamic acid (10 mg/ml in 50% ACN, 0.1% TFA). The mixture is spotted on the MALDI target and allowed to air dry. The sample is analyzed by a Microflex-LRF mass spectrometer (Bruker Daltonics, Billerica, MA) in positive ion reflector mode. External calibration is done using a peptide calibration mixture (4 to 6 peptides) on a spot adjacent to the sample. The raw data is processed in the FlexAnalysis software (version 3.4.7, Bruker Daltonics) and exported in mzXML format. The mzXML files were analyzed on ProteoWizard. Data points were normalized to the intensity of the undigested peptide input and plotted on SigmaPlot v11.0. The reaction was reproduced in three separate experiments.

The peptides used in this study include LL-37, its truncated variant LL-31, LFchimera and IDR-1018. All peptides were synthesized using Fmoc-protected amino acids (Orpegen Pharma GmbH, Heidelberg, Germany) with a Syro II peptide synthesizer (Biotage, Uppsala, Sweden) and purified with an Ultimate 3000 RP-HPLC (Thermo Scientific, MA) to a purity of at least 95% as previously described [15 (link)]. The authenticity of the peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) on a Microflex LRF mass spectrometer equipped with an additional gridless reflection (Bruker Daltonik, Bremen, Germany) as described previously [15 (link)]. Amino acid sequences and characteristics of the peptides investigated are shown in Table 1. The antibiotics used were minocycline hydrochloride (Sigma-Aldrich, St. Louis MO) and doxycycline (Sigma-Aldrich, St. Louis MO); two drugs commonly used as adjunctive treatment for periodontitis.