Easy spray nanoesi source

Tissue sample homogenization and sonication were carried out by means of the Wheaton® 903475 Overhead Stirrer apparatus (Wheaton, Millville, New Jersey, USA) and Branson Sonifier 450 (Branson Ultrasonics, Danbury, USA), respectively. Total protein concentration was determined in duplicate by Bradford assay (Bio-Rad Laboratories, Hercules, California, USA) by means of a UV-Vis spectrophotometer (8453 UV-Vis Supplies, Agilent Technologies, Waldbronn, Germany) using BSA as the protein of reference. HPLC-ESI-MS/MS analyses were performed on an UltiMate 3000 RSLCnano System coupled to an Orbitrap Elite MS detector with EASY-Spray nanoESI source (Thermo Fisher Scientific). EASY-Spray columns 15 cm × 50 μm ID, PepMap C18 (2 μm particles, 100 Å pore size), and 15 cm × 75 μm ID, PepMap C18 (5 μm particles, 300 Å pore size) (Thermo Fisher Scientific), were used for bottom-up and top-down analyses, respectively, in coupling to an Acclaim PepMap 100 cartridge (C18, 5 μm, 100 Å, 300 μm i.d. × 5 mm) (Thermo Fisher Scientific).

Protein A/G PLUS-Agarose, mouse monoclonal Ab against cystatin D-C₂₆, HRP conjugated anti-mouse secondary Ab, and nonspecific primary Ab were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Chemicals and reagent for SDS-PAGE and Western blot were purchased from Bio-Rad (Hercules, CA, USA), such as the ChemiDoc MP Imaging System. All the chemicals and reagents used for in-gel tryptic digestion and mass spectrometry analyses and the kit for the bicinchoninic acid (BCA) assay were purchased from MERCK-Sigma-Aldrich (Darmastadt, Germany), such as the NanoDrop 2000 Spectrophotometer used for determination of the protein concentration. HPLC-high-resolution ESI-MS and MS/MS experiments were carried out using an Ultimate 3000 nano-HPLC apparatus (Dionex, Sunnyvale, CA, USA) coupled through an EASY-spray nano ESI source (Thermo Fisher Scientific, San Jose, CA, USA) the LTQ-Orbitrap Elite mass spectrometer. The trap column (5 mm × 300 µm I.d.) and the EASY-spray^TM C18 nanoHPLC column (150 mm × 50 µm, particle size of 2 µm) were purchased from Thermo Fisher Scientific.

The RNA sequencing and hepatic protein quantification were previously described in detail [23 (link)]. Illumina A TruSeq Stranded mRNA kit (Illumina, Inc., San Diego, CA, USA) was used to prepare RNA sequencing libraries. Sequencing was carried by using the Illumina NextSeq 500 platform (Illumina, Inc., San Diego, CA, USA). Raw sequencing data can be downloaded from the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress; last accessed 26 May 2022), accession number E-MTAB-8032. EdgeR was used to perform pairwise gene comparisons, with all genes with a count per million value of more than one in six. After removing low-count genes, there were 15,134 genes for analysis. The p-values were adjusted by using the Benjamini–Hochberg method [37 (link)], with a false discovery rate (FDR) set at q < 0.05.
A Q Exactive Plus hybrid quadrupole Orbitrap mass spectrometer fitted with an EASY-Spray nano-ESI source (Thermo Fisher Scientific, Wilmington, DE, USA) was used to identify and quantify hepatic proteins. Limma was used to perform pairwise protein comparisons for those proteins that yielded normalised intensities in at least 75% of the compared samples. The p-values were adjusted by using the Benjamini–Hochberg method. Mass spectrometry proteomics data were added to the ProteomeXchange Consortium via the PRIDE140 partner repository, with the dataset identifier PXD014050.