Protein extraction. Larval brains were dissected in PBS, transferred to a 1.5-ml tube, and frozen in liquid nitrogen after removing the buffer. One hundred fifty brains of each genotype were homogenized in 150 μl of a buffer containing 4% SDS, 100 mM tris-HCl (pH 7.6), and 0.1 M dithiothreitol and incubated at 95°C for 3 min. The samples were sonicated to shear the DNA to reduce the viscosity of the sample. Before starting sample processing, the lysate was clarified by centrifugation at 16,000g for 5 min.

Sample preparation, TMT labeling, and basic reversed-phase prefractionation. Protein extracts were quantified using the Pierce 660 Protein Assay Kit (#22662) and Ionic Detergent Compatibility Reagent (#22663). They were alkylated with 2-iodoacetamide and digested with trypsin following the Filter Aided Sample Preparation (FASP) protocol (49). After digestion and requantification at the peptide level by the Colorimetric Peptide Assay (Pierce Thermo, #23275), samples were isotopically labeled with the corresponding TMT10plex reagent (Thermo Fisher Scientific) according to the experimental design (labels 126 to 131). Validation for correct isotopic labeling was performed by LC-MS/MS, and samples were then mixed in two different batches (taking into account peptide quantification) and desalted using PolyLC C18 and PolyLC SCX strong cationic exchange tips. Each of the two TMT10plex experiments was fractionated offline by high-pH reversed-phase peptide chromatography using Pierce columns (ref. 84868). Ten fractions were collected for each batch (F0 to F9), dried, and reconstituted in 1% formic acid, 3% acetonitrile for nanoLC-ESI-MS/MS analysis (600 ng of protein on column).

Nanoliquid chromatography electrospray ionization tandem mass spectrometry. Peptides from basic reversed-phase prefractionation (20 fractions from two TMT10plex experiments) were analyzed using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) equipped with a Thermo Scientific Dionex Ultimate 3000 ultrahigh-pressure chromatographic system (Thermo Fisher Scientific) and Advion TriVersa NanoMate (Advion Biosciences Inc.) as the nanospray interface. Peptide mixtures were loaded to a μ-precolumn (300 μm inside diameter × 5 mm, C18 PepMap100, 5 μm, 100 Å, C18 Trap column; Thermo Fisher Scientific) at a flow rate of 15 μl/min and separated using a C18 analytical column (Acclaim PepMap TM RSLC: 75 μm × 75 cm, C18 2 μm, nanoViper) with a flow rate of 200 nl/min and a 300-min run, comprising three consecutive steps with linear gradients from 1 to 35% B in 262 min, from 35 to 50% B in 5 min, and from 50 to 85% B in 2 min, followed by isocratic elution at 85% B in 5 min and stabilization to initial conditions (A = 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile).

The mass spectrometer was operated in a data-dependent acquisition mode. In each data collection cycle, one full MS scan (400 to 1600 m/z) was acquired in the Orbitrap [1.2 × 105 resolution setting and automatic gain control (AGC) of 2 × 105]. The following MS2-MS3 analysis was conducted with a top speed approach. The most abundant ions were selected for fragmentation by collision-induced dissociation (CID). CID was performed with a collision energy of 35%, 0.25 activation Q, an AGC target of 1 × 104, an isolation window of 0.7 Da, a maximum ion accumulation time of 50 ms, and turbo ion scan rate. Previously analyzed precursor ions were dynamically excluded for 30 s. For the MS3 analyses for TMT quantification, multiple fragment ions from the previous MS2 scan (SPS ions; synchronous precursor selection) were co-selected and fragmented by HCD using a 65% collision energy and a precursor isolation window of 2 Da. Reporter ions were detected using the Orbitrap with a resolution of 60,000, an AGC of 1 × 105, and a maximum ion accumulation time of 120 ms.

Spray voltage in the NanoMate source was set to 1.60 kV. Radio frequency lenses were tuned to 30%. The spectrometer was working in positive polarity mode, and singly charge state precursors were rejected for fragmentation.

Database search. Database searches were performed with Proteome Discoverer v2.1.0.81 software (Thermo Fisher Scientific) using Sequest HT search engine and UniProt Canonical and Isoforms DROME_2017_06 with contaminants. Search was run against targeted and decoy database to determine the false discovery rate (FDR). Search parameters included trypsin, allowing for two missed cleavage sites, carbamidomethyl in cysteine and TMT peptide N terminus as static modification and TMT in K, methionine oxidation, and acetylation in protein N terminus as dynamic modifications. Peptide mass tolerance was 10 parts per million (ppm), and the MS/MS tolerance was 0.6 Da in MS2 and 20 ppm in MS3. Peptides with a q value lower than 0.1 and an FDR of <1% were considered as positive identifications with a high confidence level.

Quantitative analysis. TMT reporter ion intensities were used for protein quantification. Unique peptides (peptides that are not shared between different protein groups) were considered for further quantitative and statistical analysis. Within each TMT experiment, peptide quantitation was normalized by summing the abundance values for each channel over all peptides identified within an experiment, and then the channel with the highest total abundance was taken as a reference and all abundance values were corrected in all other channels by a constant factor per channel so that, at the end, the total abundance is the same for all channels. Protein quantitation was done by summing all peptide normalized intensities for a given protein. Protein intensities were scaled so that, for every protein in an experiment, the average of all channels is 100. Proteins were only considered quantifiable if all quan channels have abundance values.

DanteR (50), by Pacific Northwest National Laboratory, was used to preprocess, visualize data (boxplots and principal components analysis), and perform relative quantification of proteins labeled with TMT. Protein quantitative measurements were log2-transformed, and normalization across the four TMT10plex experiments was performed using quantile normalization (51). Two-way analysis of variance (ANOVA) was performed at the protein level using a linear model. Conditions were considered as the principal factor and TMT batch as the second factor. Weighting function was used to allow data variability to depend on data value. Comparisons considering condition or age were performed. Last, P values were adjusted for multiple testing using the Benjamini-Hochberg FDR correction. Data were also processed by performing a one-way ANOVA statistical analysis to take also into account those proteins found only in one batch. Differential expressed proteins were determined using an adjusted P value cutoff of 0.05 and a fold change lower than 0.67 (down) or higher than 1.5 (up).

Venn diagrams. Venn diagrams were done using the web application BioVenn ( (52).

GO analysis. Functional annotation of GO terms was performed using the online tool Database for Annotation, Visualization and Integrated Discovery (DAVID 6.8; GO terms for biological process (GOTERM_BP_DIRECT) with a P value of <0.05 were accepted as a significant enrichment.

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