Tandem mass tag–mass spectroscopy (TMT-MS)

An 11-plex pool of TMT labeled peptide samples was prepared as previously described, with a slight modification to the method [20]. Briefly, five wild-type and six Lrrc6 KO mouse testes were homogenized using 8 M urea, then reduced using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and alkylated using iodoacetamide (IAA). The proteins were centrifuged at 21,000 g to remove pellets. After the buffer of the supernatants was exchanged, protein concentration was quantified by measuring the absorbance at 205 nm on the NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific, MA, USA) and adjusted to 2.2 μg/μL. The protein samples were digested with trypsin at 37 °C overnight at 1:50 trypsin ratio. The tryptic peptide samples were labeled with TMT11 based on a TMT/protein ratio (w/w) of 1:1 for 30 min, and an equal amount of each labeled sample was pooled. The pooled peptide samples of 11-plex were fractionated via basic reversed-phase liquid chromatography using a 50-cm C18 capillary column. The peptides were eluted in a 52-min gradient, and 42 fractions were collected every minute and concatenated into 10 fractions. Peptides were detected using Orbitrap Fusion Lumos, as previously described [21]. The raw-files were imported to IP2 pipeline, which searched the generated MS data against the UniProt Mus musculus database downloaded from the European Bioinformatics Institute website (https://www.uniprot.org/proteomes/UP000000589) and filtered the data using the default parameters [22].