Analyst qs 2

For intact fly analysis, individual animals were attached to a glass cover slip using adhesive pads (G304, Plano, Wetzlar, Germany). The cover slips were mounted on a custom-milled sample holder containing a rectangular, 1.8 mm deep well. Sample height was adjusted by choosing a stack of 0.2 mm-thick adhesive pads (G3347, Plano). Mass spectra were generated using a prototype orthogonal-extracting mass spectrometer (oTOF-MS) as described previously (Yew et al., 2011 (link)). The oTOF-MS was equipped with a modified oMALDI2 ion source (AB Sciex, Concord, ON, Canada) and an N₂ laser (λ = 337 nm) operated at a pulse repetition rate of 30 Hz. N₂ was used as buffer gas at p = 2 mbar. This elevated pressure is critical to achieve an efficient collisional cooling environment for generation of weakly bound [M + K]⁺ ions that constituted the major molecular ion species. Before starting the actual measurements, external mass calibration was achieved with red phosphorus, resulting in a mass accuracy of approximately 25 ppm. Approximately 900 laser shots were placed at one position to achieve a mass spectrum (30 s @30 Hz). All spectra were acquired in positive ion mode and processed using MS Analyst software (Analyst QS 2.0, AB Sciex, Concord, ON, Canada).

A 1200 series HPLC (Agilent Technologies Inc., Santa Clara, CA, USA) was used in combination with a QSTAR^TM Elite QTOF-MS (AB SCIEX, Middlesex, MA, USA) for urine metabolic profile measurements. The urine analysis method that was established in our previous study was applied with other parameters detailed in the Supporting Information. Data acquisition was performed with Analyst QS 2.0 (AB SCIEX, Middlesex, MA, USA), and qualitative analysis was performed using PeakView 2.0 (AB SCIEX, Middlesex, MA, USA) equipped with Formula Finder, which was directly linked to the ChemSpider database (Royal Society of Chemistry, Cambridge, UK).

Raw data were collected by Analyst QS 2.0 controlling software (AB Sciex), and Maxquant (version 1.5.2.8, Max Planck Institute of Biochemistry, Martinsried, Germany) was used to identify the proteins in a search of the protein database of Hemiptera downloaded from National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). Parameters for protein identification were as follows: MS/MS tol. (FTMS) = 20 ppm, MS/MS tol (ITMS) = 0.5 Da, oxidation (M), FDR ≤ 0.01. The significantly different ratio was set at 1.2-fold: proteins were considered as upregulated if the ratio was >1.2 and downregulated if the ratio was <0.83.