4.4.1. Isolation and Identification of Palmitoyl-Proteins

Acyl-biotinyl exchange chemistry (ABE) technology [15] was used to purify the palmitoyl-proteins, according to the protocol described by Wan et al. [16], which we have applied with minor modifications to bovine follicular cell proteins. Detailed protocol is reported in Supplementary Materials, protocol “ABE isolation and identification of palmitoyl-proteins”.

Briefly, total proteins were extracted from frozen pellets of GC (total amount is about 0.5 g), and COCs (n = 500), collected from about 120 ovaries in tree experiments, using 10 mM N-ethylmaleimide (NEM) to block the free thiols. After removing the NEM, the subsequent treatments with hydroxylamine (HA) were performed in order to cleave the palmitoylation thioester linkages. Following treatment with HPDP-biotin biotinylated free thiols. Proteins were precipitated three times using chloroform-methanol, resuspended in 4% SDS and divided into two equal portions (+HA sample and −HA sample as control). The +HA sample was diluted five-fold by addition hydroxylamine-containing +HA buffer (0.7 M HA, 1 mM HPDP–biotin, 0.2% Triton X-100, 1 mM PMSF, 1x PI pH 7.4); for the −HA samples, −HA buffer (50 mM Tris, 1 mM HPDP–biotin, 0.2% Triton X-100, 1 mM PMSF, 1x PI, pH-7.4) was added. The +HA and −HA samples were incubated at room temperature for 1 h with end-over-end rotation, and then precipitated to remove chemicals. After serial precipitations, affinity purification of biotinylated proteins was performed per each −HA and +HA sample, then precipitated using 100% trichloroacetic acid (TCA), and stored −20 °C.

For identification of palmitoylated proteins, each pellet from +HA and −HA samples was resuspended in SDS-PAGE sample buffer containing 4% SDS, 5% beta-mercaptoethanol, 125 mM Tris-HCl pH 6.8, 20% glycerol and bromophenol blue as the dye, and incubated for 5 min at 95  °C, then centrifuged at 10,000× g for 5 min, and SDS-PAGE was performed. For exhaustive identification, each line of −HA and +HA samples were sectioned into 20 slices which then used for in-gel digestion and nano-LC-MS/MS analysis performed using a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to an Ultimate^® 3000 RSLC Ultra High Pressure Liquid Chromatographer (Dionex, Amsterdam, The Netherlands).

For protein identification, mass spectrum ion searches were performed using Mascot search engine v2.3.2 (Matrix Science, London, UK) via Proteome Discoverer 2.1 software (ThermoFisher Scientific, Bremen, Germany). Research was performed against the mammalian non-redundant NCBI database (released July 2018). Peptides and proteins identified by MASCOT were subjected to Scaffold v4.8.4 software (Proteome Software Inc., Portland, OR, USA). Peptide and protein identifications were accepted if they could be established at greater than 95% probability by the Peptide Prophet algorithms [88,89], respectively. Proteins sharing significant peptide evidence were grouped into clusters. For each protein identified to be non-bovine, a manual blast analysis against Bos taurus protein databases was performed to obtain a species-specific identification of the protein. The abundance of identified proteins within one sample was estimated by calculating the Exponentially Modified Protein Abundance Index [90].

To characterize enriched palmitoylated proteins, +HA and −HA conditions analyzed in triplicate (R1, R2, R3), with three technical replicates per band were compared using Scaffold Q+ software (v4.4, Proteome Software, Portland, OR, USA). Label-free quantitative approach was employed. Protein clusters were defined as enriched palmitoylated proteins if they met the following conditions: (1) detection once among the three replicates only in +HA and never in control condition −HA; (2) detection in both conditions but with an enrichment in +HA with a fold change > 3. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [91] partner repository with the dataset identifiers PXD020540 and PXD020547.