Affinity purification mass spectrometry

Roughly 600 embryos for each sample were collected from overnight cages kept at 25°C for the following crosses: yw (control) or females ; 67-GAL4/+; UASt-Toll-8::sYFP2/+; x males ; 67-GAL4/+; UASt-Toll-8::sYFP2/+; (Toll-8::YFP maternal and zygotic overexpression), dechorionated with bleach, transferred directly to lysate buffer (10 mM Tris/Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA; 0.5% NP-40, supplemented with protease and phosphatase inhibitors) and crushed manually on ice over 30 minutes. Lysates were centrifuged to clear debris and protein concentrations of post-centrifugation supernatants were determined. The crude protein yield per lysate sample is usually 1000∼3000 μg. In each experiment, lysates of comparable protein concentration were incubated with pre-rinsed GFP nano-trap agarose resin (Chromotek, gta-20) at 4°C for 90 min, rinsed 3 x and resuspended in 2x Laemmli buffer with DTT. Protein extraction and purification was performed 3 times each for each cross and verified with silver staining. Protein samples were further purified on NuPAGE 4-12% Bis-Tris acrylamide gels (Life Technologies) and treated with in-gel trypsin digestion (Shevchenko et al., 1996) with minor modifications. Peptides were harvested with two extractions, first in 5% formic acid and then in 5% formic acid in 60% acetonitrile. Samples were reconstituted with 0.1% trifluoroacetic acid in 4% acetonitrile and analyzed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) with an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Electron, Bremen, Germany) online with an Ultimate 3000RSLCnano chromatography system (Thermo Fisher Scientific, Sunnyvale, CA). A detailed mass spectrometry protocol is available upon request.

Relative intensity-based label-free quantification (LFQ) was processed using the MaxLFQ algorithm (Cox et al., 2014) from the freely available MaxQuant computational proteomics platform (Cox and Mann, 2008). Spectra were searched against a Drosophila melanogaster database (UniProt Proteome reference, date 2017.08; 21982 entries). The false discovery rate (FDR) at the peptide and protein levels were set to 1% and determined by searching a reverse database. For protein grouping, all proteins that could not be distinguished based on their identified peptides were assembled into a single entry according to the MaxQuant rules. The statistical analysis was done with Perseus program (version 1.5.1.6) from the MaxQuant environment (www.maxquant.org). Quantifiable proteins were defined as those detected in at least 67% of samples in at least one condition. Protein LFQ normalized intensities were base 2 logarithmized to obtain a normal distribution. Missing values were replaced using data imputation by randomly selecting from a normal distribution centered on the lower edge of the intensity values that simulates signals of low abundant proteins using default parameters (a downshift of 1.8 standard deviation and a width of 0.3 of the original distribution). To determine whether a given detected protein was specifically differential, a two-sample t-test was done using permutation-based false discovery rate (pFDR) with a threshold at 0.1% (5000 permutations). The p-value was adjusted using a scaling factor s0=1 (Table S1). In Figure 3, differential proteins are highlighted by a cut-off for log₂|Fold change|>2 and a p-value<0.01. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Vizcaíno et al., 2014) partner repository with the dataset identifier PXD017895.