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+<p><strong>Sample preparation for proteomic analysis</strong></p>

+

+<p>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&nbsp;<em>g</em>, for 20 min at 4&nbsp;<sup>o</sup>C and the supernatant fractions were taken. Protein concentrations were determined using a Pierce BCA Assay Kit (Thermo Fisher Scientific, USA), according to manufacturer&rsquo;s instructions. Samples were prepared for liquid chromatography tandem mass spectrometry (LC-M/MS) using the Phase Transfer Surfactant Method (Masuda&nbsp;<em>et al</em>., 2008; Masuda&nbsp;<em>et al</em>., 2009), with minor modifications as previously described (Mostafa&nbsp;<em>et al</em>., 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.</p>

+

+<p><strong>MS measurement</strong></p>

+

+<p>Samples were measured using a Q Exactive Plus Orbitrap LC&ndash;MS/MS System (Thermo Fisher), equipped with a Nanospray Flex ion source. Peptides were separated on 3-&micro;m particle, 75-&micro;m inner diameter, 12-cm filling length C18 columns (Nikkyo Technos Co., Ltd., Japan). For each sample, 600&thinsp;ng was injected and the samples were measured with data-independent acquisition (DIA).&nbsp;&nbsp;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&nbsp;<em>et al</em>., 2020), except for DDA the first mass for MS2 scans was not fixed and for DIA the AGC target of DIA segments was 3e<sup>6</sup>.</p>

+

+<p><strong>Protein identification and quantification</strong></p>

+

+<p>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&nbsp;<em>et al</em>., 2020). FDR was estimated with the mProphet approach (Reiter&nbsp;<em>et al</em>., 2011) and set to 0.01 at both peptide precursor level and protein level (Rosenberger&nbsp;<em>et al</em>., 2017).&nbsp;</p>

+

+<p><strong>References</strong></p>

+

+<p>Masuda, T., Tomita, M., and Ishihama, Y. (2008) Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis.&nbsp;<em>J. Proteome Res</em>.&nbsp;<strong>7</strong>, 731-740.</p>

+

+<p>Masuda, T., Saito, N., Tomita, M., and Ishihama, Y. (2009) Unbiased quantification of&nbsp;<em>Escherichia coli&nbsp;</em>membrane proteome using phase transfer surfactants.&nbsp;<em>Mol. Cell. Proteomics</em>&nbsp;<strong>8</strong>, 2770-2777.</p>

+

+<p>Mostafa, D., Yanagiya, A., Georgiadou, E., Wu, Y., Stylianides, T., Rutter, G. A., Suzuki, T., &amp; Yamamoto, T. (2020). Loss of &beta;-cell identity and diabetic phenotype in mice caused by disruption of CNOT3-dependent mRNA deadenylation.&nbsp;<em>Commun. Biol.</em>&nbsp;<strong>3</strong>, 476.</p>

+

+<p>Reiter, L., Rinner, O., Picotti, P., H&uuml;ttenhain, R., Beck, M., Hengartner, M. O., and Aebersold, R. (2011) mProphet: automated data processing and statistical validation for large scale SRM experiments.&nbsp;<em>Nat. Methods</em>&nbsp;<strong>8</strong>, 430&ndash;435.</p>

+

+<p>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., R&ouml;st, 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.&nbsp;<em>Nat. Methods</em>&nbsp;<strong>14</strong>, 921&ndash;927.</p>

diff --git a/general/datasets/Riken_wu_bxd_cdhf_wadip_0920/specifics.rtf b/general/datasets/Riken_wu_bxd_cdhf_wadip_0920/specifics.rtf
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+Riken-Wu BXD CD-HF White Adipose Proteome (Sep20)
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