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Experiment design
Sample preparation for proteomic analysis
Proteins were extracted from adipose tissue samples in lysis buffer containing 12 mM sodium deoxycholate, 12 mM sodium N-dodecanoylsarcosinate, and 100 mM Tris.Cl pH 9.0, with cOmplete, mini, EDTA-free Protease Inhibitor Cocktail (Roche, Switzerland), using homogenization with a glass dounce followed by sonication for 15 minutes using a Bioruptor water bath sonicator on high power, with cycles of one min sonication followed by one min rest (Cosmo Bio Co. Ltd., Japan). The samples were centrifuged at 18,000 x g, for 20 min at 4 oC and the supernatant fractions were taken. Protein concentrations were determined using a Pierce BCA Assay Kit (Thermo Fisher Scientific, USA), according to manufacturer’s instructions. Samples were prepared for liquid chromatography tandem mass spectrometry (LC-M/MS) using the Phase Transfer Surfactant Method (Masuda et al., 2008; Masuda et al., 2009), with minor modifications as previously described (Mostafa et al., 2020). Afterwards, dried Lys-C/tryptic peptides were dissolved in 0.1% TFA and desalted using MonoSpin C18 columns (GL Sciences Inc., Japan). Peptides were eluted from C18 columns in 0.1% (v/v) TFA in 50% (v/v) acetonitrile and dried in a centrifugal vacuum concentrator. Tryptic peptides were dissolved in 0.1% (v/v) formic acid, 3% (v/v) acetonitrile in water for MS analysis and the peptide concentrations were determined using a Pierce Quantitative Colorimetric Peptide Assay Kit (Thermo Fisher). A portion of the peptides from the samples were pooled and fractionated using a Pierce High pH Reversed-Phase (HPRP) Peptide Fractionation Kit (Thermo Fisher) to generate a spectral library.
MS measurement
Samples were measured using a Q Exactive Plus Orbitrap LC–MS/MS System (Thermo Fisher), equipped with a Nanospray Flex ion source. Peptides were separated on 3-µm particle, 75-µm inner diameter, 12-cm filling length C18 columns (Nikkyo Technos Co., Ltd., Japan). For each sample, 600 ng was injected and the samples were measured with data-independent acquisition (DIA). For HPRP fractions, 450 ng was injected and the samples were measured with data-dependent acquisition (DDA). LC-MS/MS conditions were as previously described (Mostafa et al., 2020), except for DDA the first mass for MS2 scans was not fixed and for DIA the AGC target of DIA segments was 3e6.
Protein identification and quantification
Raw files from DDA measurements were searched against a mouse-specific database (uniprot-reviewed_Mus_musculus_10090_.fasta) using Proteome Discoverer v2.4 software (Thermo Fisher). Filtered output was used to generate a sample-specific spectral library using Spectronaut software (Biognosys, Switzerland). Raw files from DIA measurements were used for quantitative data extraction with the generated spectral library, as previously described (Mostafa et al., 2020). FDR was estimated with the mProphet approach (Reiter et al., 2011) and set to 0.01 at both peptide precursor level and protein level (Rosenberger et al., 2017).
References
Masuda, T., Tomita, M., and Ishihama, Y. (2008) Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J. Proteome Res. 7, 731-740.
Masuda, T., Saito, N., Tomita, M., and Ishihama, Y. (2009) Unbiased quantification of Escherichia coli membrane proteome using phase transfer surfactants. Mol. Cell. Proteomics 8, 2770-2777.
Mostafa, D., Yanagiya, A., Georgiadou, E., Wu, Y., Stylianides, T., Rutter, G. A., Suzuki, T., & Yamamoto, T. (2020). Loss of β-cell identity and diabetic phenotype in mice caused by disruption of CNOT3-dependent mRNA deadenylation. Commun. Biol. 3, 476.
Reiter, L., Rinner, O., Picotti, P., Hüttenhain, R., Beck, M., Hengartner, M. O., and Aebersold, R. (2011) mProphet: automated data processing and statistical validation for large scale SRM experiments. Nat. Methods 8, 430–435.
Rosenberger, G., Bludau, I., Schmitt, U., Heusel, M., Hunter, C. L., Liu, Y., MacCoss, M. J., MacLean, B. X., Nesvizhskii, A. I., Pedrioli, P. G. A., Reiter, L., Röst, H. L., Tate, S., Ting, Y. S., Collins, B. C., and Aebersold, R. (2017) Statistical control of peptide and protein error rates in large-scale targeted data-independent acquisition analyses. Nat. Methods 14, 921–927.