Autoflex 3 smartbeam maldi tof mass spectrometer

Samples were prepared in 2 mg/mL water solution and filtered with a 0.22 μm syringe filter (Pall, Ann Albor, MI, USA). Then, 1 μL of sample was mixed with the same volume of 2,5-dihydroxybenzoic acid (Sigma-Aldrich, St. Louis, MO, USA) as a sample matrix and air-dried for MALDI-TOF analysis. The analysis was performed on an Autoflex III Smart Beam MALDI-TOF mass spectrometer (Bruker, Bremen, Germany) in the positive ion mode. For ¹H NMR spectral analysis, samples were dissolved in D₂O (30 mg/mL), and the spectra were carried out on a Bruker AV 500 MHz (Bruker, Karlsruhe, Germany).

To obtain MFPs by MALDI mass spectrometry (MALDI BeeTyping^®), haemolymph samples were handled according to the protocol published by Arafah et al. (2019) with modifications to optimise sample analysis [63 (link)]. Each individual haemolymph sample was analysed with an AutoFlex III Smartbeam^® MALDI-TOF mass spectrometer (Bruker Daltonics, Germany). MFPs were acquired following the Bruker BioTyper^® recommendations (matrix, method of sample deposition, and detection) with minor adjustments. Briefly, the haemolymph samples were diluted 1:10 in water acidified with 1% TFA. A volume of 1 µL from each diluted sample was spotted on a MALDI MTP 384 polished ground steel plate (Bruker Daltonics), dried under gentle vacuum, and then mixed with 1 μL of 4-HCCA. Following co-crystallisation of the haemolymph spots with the matrix droplet, MALDI MFPs were recorded in a linear positive mode and in an automatic data acquisition using FlexControl software v3.4 (Bruker Daltonics). The samples were manually spotted in triplicate, each of the three spots being read three times.