Nano-liquid chromatography mass spectrometry (nLC-MS/MS)

The details of nLC-MS/MS analysis has been described elsewhere⁴⁴ and used with a minor modifications for PTM analysis.

Protein digestion

The silver stained protein spots in gels were excised carefully with help of a sterilized spatula and transferred into separate vials. Destaining and acetone precipitation was carried out. In-gel digestion was performed; samples were reduced with 5 mM TCEP (Tris 2-Carboxyethyl Phosphine) at 55°C for 1 hour and further alkylated with 50 mM iodoacetamide for 30 min at room temperature in the dark. The gel spots were shrinked with acetonitrile and air-dried for few minutes at room temperature followed by digestion with trypsin (1:50, trypsin/lysate ratio) for 16 hours at 37°C. Digests were dried using speed vac for 1 hour and pellet was dissolved in buffer A (5% acetonitrile, 0.1% formic acid). Digests were cleaned by Sep-Pak. Titanium method was performed for phospho binding. Digests were cleaned up again using C18 silica cartridge (The Nest Group, Southborough, MA) following manufacturer’s protocol and dried using speed vac. The desalted dried pellet was reconstituted in buffer A (5% acetonitrile, 0.1% formic acid).

Liquid chromatography mass spectrometry analysis

All the experiments were performed using EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to Q Exactive mass spectrometer (Thermo Fisher Scientific, Germany) equipped with nano electrospray ion source. Peptide mixture (1.0 µg) was loaded on a precolumn and was resolved using 5 cm PicoFrit column (360 µm outer diameter, 75 µm inner diameter, 10 µm tip) filled with 1.9 µm of C18-resin (Dr Maeisch, Germany). The peptides were loaded with buffer A and eluted with a 0–40% gradient of buffer B (95% acetonitrile, 0.1% formic acid) at a flow rate of 500 nl/min for 10 min. The QExactive was operated using the Top10 HCD data-dependent acquisition mode with a full scan resolution of 70,000 at m/z 400. MS/MS scans were acquired at a resolution of 17500 at m/z 400. Lock mass option was enabled for polydimethylcyclosiloxane (PCM) ions (m/z = 445.120025) for internal recalibration during the run. MS/MS data was acquired using a data-dependent top 10 method dynamically choosing the most abundant precursor ions from the survey scan.

Protein identification and PTM analysis

The .raw files generated were analyzed using Proteome Discoverer (v2.2) against the in-house Uniprot reference proteome database (Capra hircus, Ovis aries, Homo sapiens and Bos taurus). Due to scarcity of available protein annotations for goat, the analysis was carried out with goat, human, sheep and cow database with 7056, 20117, 27666, and 23869 entries respectively. For Sequest search, the precursor and fragment mass tolerances were set at 10–15 ppm and 0.5 Da, respectively. The protease used to generate peptides, that is the enzyme specificity was set for trypsin/P (cleavage at the C terminus of “K/R”: unless followed by “P”) along with maximum missed cleavages value of 2. Carbamidomethyl on cysteine as fixed modification and oxidation of methionine and N-terminal acetylation and phosphorylation (S, T, Y, D) were considered as variable modifications for database search. Peptide spectrum match and protein false discovery rate (FDR) were set to 0.01.