Spermathecal fluid proteins were cleaned up via acetone precipitation overnight at 20 C. The precipitated protein was pelleted and washed twice with 500 ul of 80% acetone, then the pellet was allowed to air dry (ca. 5 min) prior to solubilization in 50 ul of digestion buffer (6 M urea, 2 M thiourea).
Approximately 25 ug of protein were reduced (0.5 ug dithiothreitol, 20 min), alkylated (2.5 ug iodoacetamide, 30 min, dark), and digested (0.5 ug Lys-C for 3 h, then 0.5 ug trypsin overnight). Digested peptides were acidified with one volume of 1% trifluoroacetic acid and desalted with high-capacity STAGE tips. Eluted samples were dried (SpeedVac, Eppendorf, 45 min) and resuspended in Buffer A (0.1% formic acid). Peptide concentrations were determined using a NanoDrop (Thermo, 280 nm) and sample orders were randomized for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
Peptides (0.5 ug for each sample) were injected on an EASY-nLC 1000 liquid chromatography system (Thermo) coupled to an Impact II Q-TOF mass spectrometer (Bruker). The LC system included a fused-silica (5 um Aqua C18 particles (Phenomenex)) fritted 2 cm trap column connected to a 50 cm analytical column packed with ReproSil C18 (3 um C18 particles (Dr. Maisch)). The separation gradient ran from 5% to 35% Buffer B (80% acetonitrile, 0.1% formic acid) over 120 min, followed by a 15 min wash at 95% Buffer B (flow rate: 250 uL/min). The instrument parameters were: scan from 150 to 2200 m/z, 100 us transient time, 10 us prepulse storage, 7 eV collision energy, 1500 Vpp Collision RF, a +2 default charge state, 18 Hz spectral acquisition rate, 3.0 s cycle time, and the intensity threshold was 250 counts.
Mass spectrometry data were searched using MaxQuant (v1.6.1.0) using default parameters, except match between runs was enabled. Peptide spectral matches, peptide identifications and protein identifications were controlled at 1% false discovery rates (FDRs). Data were analyzed in Perseus (v1.6.1.1). Proteins differentially expressed between heat-shocked and non-heat-shocked samples, as well as among tissues (semen, virgin spermathecae, and mated spermathecae), were identified using t-tests and significant results were corrected to either 5% (heat-shock and cold-shock) or 10% FDR (pesticide stress) (Banjamini-Hochberg method).
[doi:10.25345/C55T23]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: Apis mellifera ; Honey bee ; Queen ; Spermatheca
Principal Investigators: (in alphabetical order) |
Leonard Foster, University of British Columbia, Canada |
Submitting User: | amcafee |
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