Autoflex mass spectrometer

Each peptide mixture (0.5 µL) was spotted onto a MALDI target and overlaid with 0.5 µL of matrix (5 mg/mL 2-cyano-4-hydroxycinnamic acid dissolved in 50% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid) using the dry droplet technique. Positive ion mass spectra were acquired over a 600–3000 m/z range on an Autoflex mass spectrometer (Bruker, Bremen, Germany) and manually evaluated using flexAnalysis 3.3 software (Bruker Daltonics, Bremen, Germany).
For known proteins, theoretical masses of the peptides containing one or more methionine residues or methionine analogs were generated using GPMAW software 7.1 [27 (link)]. Unknown proteins were identified by peptide mass fingerprinting [28 (link)]. Monoisotopic peptide masses were matched against the SWISSPROT non-redundant database; mass tolerance was set to 20 ppm, proteins were restricted to E. coli, allowing one missed cleavage and cysteine alkylation and methionine oxidation were set as fixed and variable modifications, respectively. The identity of unknown proteins was confirmed by tandem mass spectrometry with CID fragmentation of selected peptide precursors.

Paw tissue from treated animals (see below) was shock-frozen after dissection from bone and skin followed by homogenization in a mixture of chloroform, methanol and water (2:1:1 volume) with a lab mixer (IKA® RW 14, IKW®-Werke, Staufen, Germany). After centrifugation (1500 g, 4 °C, 30 min), the chloroform (lower) phase was recovered, evaporated under nitrogen and stored at −20 °C. MALDI-TOF MS was performed immediately after extraction using 2,5-dihydroxybenzoic acid as matrix, as described earlier^{82 (link)}. Briefly, chloroform extracts or reagents (OxPAPC, PAPC; CFA)^{83 (link)} were re-dissolved in 20–30 µl of a 2,5-dihydroxybenzoic acid matrix solution (in methanol) prior to deposition onto the MALDI target. MALDI-TOF mass spectra were acquired on a Bruker Autoflex mass spectrometer, which is equipped with a pulsed nitrogen laser, emitting at 337 nm (Bruker Daltonics, Bremen, Germany). The extraction voltage was 20 kV. Gated matrix suppression was applied to prevent the saturation of the detector by matrix ions. For each mass spectrum, 128 single laser shots were averaged. The laser fluence was kept about 10% above threshold (i.e. the minimum laser fluence required to achieve detectable signals) to obtain optimum signal-to-noise (S/N) ratios. In order to enhance the spectral resolution all spectra were acquired in the reflector mode using delayed extraction conditions.

Mass spectra were recorded on a Bruker Autoflex mass spectrometer in positive linear mode with a nitrogen laser at near-threshold laser intensity. As matrix trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]-malononitrile was used. 3.5 mg matrix was dissolved in 100 μL toluene. Matrix and sample were mixed at volume ratio 1:1, and 2 μL of the mixture was applied to the MALDI plate and air-dried.

All MALDI-TOF mass spectra were acquired on an Autoflex mass spectrometer (Bruker Daltonics, Bremen, Germany) in the linear mode under delayed extraction conditions as previously described [10 (link), 21 ]. Although mass spectra recorded in the linear mode have only limited resolution and a reduced mass accuracy [21 ], the higher sensitivity and the reduced generation of fragmentation products are clear advantages of this approach. 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 the saturation of the detector by matrix ions [22 (link)].
200 single laser shots were averaged for each mass spectrum. The laser fluence was kept about five percent above threshold to obtain optimum signal to noise ratios.
Saturated 9-aminoacridine (9-AA) [23 (link), 24 ] in methanol was used as matrix for negative ion detection. Spectra were analyzed with the program FlexAnalysis.

The Toll pathway was activated in flies using heat-killed M. luteus, then incubated at 29°C for 24 h. Hemolymph was extracted as in Lindsay et al. (25 (link)), with slight modifications. Hemolymph extracted with glass capillaries from five male flies was pooled and transferred into 0.1% trifluoroacetic acid (TFA)/50% acetonitrile (ACN). One μl of each mixture was spotted on a Bruker MSP 96 ground steel plate, mixed 1:1 with a saturated solution of Universal MALDI matrix (Sigma-Aldrich) in 0.1% TFA/78% ACN, and air-dried. MALDI-TOF spectra were acquired using a Bruker Autoflex mass spectrometer. Data were collected from 1,500 to 10,000 m/z in positive linear mode, and 1,000–5,000 m/z in positive reflectron mode. Peptide calibration standard II (Bruker) was mixed with Universal MALDI matrix and used as an external calibration standard. At least ten independent samples were collected for each genotype. For peptide identification, peaks were matched to those of corresponding peaks in prior studies (13 (link), 25 (link)). Representative spectra were visualized using R 3.3.2 and ggplot2 2.2.1 (27 , 28 ).