Male C57BL/6J mice were purchased from the Jackson Laboratory Japan and were maintained under specific pathogen-free conditions on a 12 h light dark cycle. For tissue collection, mice were first euthanized by cervical dislocation and then the abdominal hair was removed with depilatory cream Veet (Reckitt Benckiser). After 30 seconds, the cream was wiped off and abdominal skin, brain (cerebrum), gastrocnemius muscle, heart, kidney, liver, lung, and thoracic aorta were collected. Collected tissues were immediately frozen in liquid nitrogen (except for the heart samples used for immunofluorescence staining). Frozen tissues were thoroughly pulverized with mortar in liquid nitrogen and kept in -80 °C until use.
Whole-tissue lysate was prepared by rotating tissue powder in 2 × Laemmli buffer (4% SDS, 20% glycerol, 0.02% bromophenol blue, 125 mM Tris-Cl, pH 6.8) for 2 h at RT. Insoluble material was removed by centrifugation (17,700 × g, 15 min, RT). To prepare low-solubility protein-enriched fraction, tissue powder was first rotated for 72 h at RT in 0.125% (heart, kidney, lung, and skin) or 0.25% (muscle) SDS/water solution supplemented with protease inhibitor cocktail (Nacalai Tesque Cat No. 04080). Pre-incubated tissues were pelleted by centrifugation (14,000 × g, 15 min, RT) and the supernatant was discarded. The pelleted tissue was resuspended in water containing protease inhibitor cocktail and rotated for 48 h at RT. Then the tissue was centrifuged again (17,700 × g, 15 min, RT). The supernatant was discarded and the pelleted tissue was resuspended and rotated in 2 × Laemmli buffer for 2 h at RT. Insoluble material was removed by centrifugation (17,700 × g, 15 min, RT) and the supernatant was used as low-solubility protein-enriched fraction. Trypsin digestion and peptide purification was performed using the S-Trap mini column (Protifi) following the manufacturer’s instructions.
Peptides were labelled with TMT16plex Isobaric Label Reagent (Thermo Fisher Scientific) according the manufacturer’s instructions. The reaction was quenched with 5% hydroxylamine for 15 min at RT. Samples were combined and cleaned up using an OASIS HLB μElution Plate (Waters). Off-line high pH reverse phase fractionation was carried out on an Agilent 1200 Infinity high-performance liquid chromatography system, equipped with a Gemini C18 column (3 μm, 110 Å, 100 × 1.0 mm, Phenomenex). An UltiMate 3000 RSLC nano LC system (Dionex) equipped with an μ-Precolumn (C18 PepMap 100, 5 μm, 300 μm i.d. × 5 mm, 100 Å) and an analytical column (nanoEase M/Z HSS T3 column 75 µm × 250 mm C18, 1.8 μm, 100 Å, Waters) was coupled directly to an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific) using the Nanospray Flex ion source in positive ion mode. Sample trapping was carried out with a constant flow of 0.05% trifluoroacetic acid in water at 30 μL/min for 6 min. Peptides were eluted from the analytical column running using solvent A (0.1% formic acid in water, 3% DMSO) with a constant flow of 0.3 μL/min in combination with an increasing percentage of solvent B (0.1% formic acid in acetonitrile, 3% DMSO). The gradient was as follows: from 2% to 8% in 4 min, from 8% to 26% in 104 min, from 28%-38% in 4 min, and finally from 38%-80% in 4 min followed by re-equilibration back to 2% B in 4 min. The peptides were introduced into the Fusion Lumos via a Pico-Tip Emitter 360 μm OD × 20 µm ID; 10 μm tip (CoAnn Technologies) and an applied spray voltage of 2.2 kV. The capillary temperature was set at 275 °C. Full mass scan was acquired with mass range 375-1500 m/z in profile mode in the Orbitrap with resolution of 120,000. The filling time was set at maximum of 50 ms with a limitation of 4 × 105 ions. Data dependent acquisition (DDA) was performed with the resolution of the Orbitrap set to 30,000, with a fill time of 94 ms and a limitation of 1 × 105 ions. A normalized collision energy of 34 was applied. MS2 data was acquired in profile mode. The acquired RAW files were searched against a Mus musculus (UP000000589, June 2020, 63854 entries) Uniprot database containing common contaminants and reversed sequences using IsobarQuant and Mascot (v2.2.07). The following modifications were included into the search parameters: Carbamidomethyl (C) and TMT16 (K) as fixed modifications, Acetyl (Protein N-term), Oxidation (M) and TMT16 (N-term) as variable modifications. For the full scan (MS1) a mass error tolerance of 10 ppm and for MS/MS (MS2) spectra of 0.02 Da was used. Further parameters were trypsin as protease with an allowance of maximum two missed cleavages; a minimum peptide length of seven amino acids; at least two unique peptides were required for a protein identification. The false discovery rate on peptide and protein level was set to 0.01. The raw output files of IsobarQuant were processed using the R programming language. Contaminants were filtered out. Log2 transformed raw TMT reporter ion intensities were normalized using vsn. Proteins were tested for differential expression using the limma package. The replicate information was added as a factor in the design matrix given as an argument to the ‘lmFit’ function of limma.
Tissue powder was homogenized with a pestle in RNAiso Plus (Takara Cat No. 9108) and the total RNA was extracted following the manufacturer's instructions. The sequencing libraries were prepared using the Illumina Stranded mRNA Prep kit (Illumina Cat No. 20040534) following the manufacturer's instructions. Paired-end sequencing (2 × 151 bp) was performed on a NovaSeq 6000 system (Illumina) using the NovaSeq 6000 S2 Reagent Kit v1.5 (300 cycles) (Illumina). Demultiplexing, quality control, and adapter trimming were performed using bcl-convert v 3.10. Poor quality reads were removed using Fastp (v 0.22.0). Clean reads were mapped to the mouse reference genome (GRCm39) using STAR (v 2.7.10b). The read counts were calculated using RSEM (v 1.3.1). Normalization and differential expression analysis were carried out using DESeq2 (v 1.36.0).