Tissues samples were obtained from ten premenopausal patients who underwent hysterectomy for symptomatic uterine leiomyomas. All procedures conformed with the Declaration of Helsinki and were approved by the Review Board of the Institute for Maternal and Child Health-IRCCS 'Burlo Garofolo' (Trieste, Italy). All subjects involved signed a written informed consent. The median age of patients was 44 years, with a range of 36-48 years. The patients were recruited from January to Febuary 2019 at the Institute for Maternal and Child Health-IRCCS 'Burlo Garofolo' (Trieste, Italy), where all hysterectomies took place. Oncologic patients, HIV, HBV, HCV-seropositive patients, and patients with adenomyosis were excluded from the study. The patients had not received hormonal therapy in the three months prior to surgery.

Two samples were collected from each patient: One from the central area of the leiomyoma and one from the unaffected myometrium. All leiomyomas were confirmed histologically as benign ordinary leiomyomas. Samples were stored at −80°C until proteomic analysis was performed.

One hundred mg of myometrium and leiomyoma tissue from ten patients were used for phosphoprotein isolation using the Phosphoprotein Enrichment kit (Thermo Fisher Scientific, Inc.). Tissues were homogenized in buffer [1% NP-40, 50 mM Tris-HCl (pH 8.0), NaCl 150 mM] with 1X Phosphatase Inhibitor Cocktail Set II (EMD Millipore), 2 mM PMSF and 1 mM benzamidine. The concentration of the supernatant was determined by Bradford assay. Tissue homogenates were then diluted to a final concetration of 0.5 mg/ml in lysis buffer provided by the Phosphoprotein Enrichment kit. Six ml of final samples were used for isolation of the phosphoproteins, according to the manufacturer's instructions.

For 2-DE analysis, a single patient-single gel strategy was adopted, and the analyzes were performed as previously described (17). For 2-DE analysis, 250 µg of proteins from each sample were denatured in 315 µl of dissolution buffer [7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 40 mM Tris, 65 mM dithiothreitol (DTT) and 0.24% Bio-Lyte 3/10 (Bio-Rad Laboratories, Inc.)] with a trace of bromophenol blue. ReadyStrip™ pH 4-7 18-cm immobilized pH gradient (IPG) strips (Bio-Rad Laboratories, Inc.) were rehydrated in dissolution buffer at 50 V for 12 h at 20°C, and isoelectric focusing (IEF) was performed in a PROTEAN IEF Cell (Bio-Rad Laboratories, Inc.). After the IEF, serial incubations were performed: first, the IPG strips were equilibrated for 15 min in an equilibration buffer [6 M urea, 2% SDS, 50 mM Tris-HCl (pH 8.8), 30% glycerol and 1% DTT] and then in another equilibration buffer containing 4% iodoacetamide instead of DTT. For the second dimension, the equilibrated IPG strips were transferred to a 12% polyacrylamide gel.

After electrophoresis, gels were fixed in 40% methanol and 10% acetic acid for 1 h, and then stained for 16 h with SYPRO Ruby (Bio-Rad Laboratories, Inc.). 2-DE gels were scanned with a Molecular Imager PharosFX System (Bio-Rad Laboratories, Inc.). Double experimental replicates were performed per sample. For all gels, molecular weights were determined by comparison with Precision Plus Protein Prestained Standards (Bio-Rad Laboratories, Inc.) covering a range 10-250 kDa, and analyzed using the Proteomweaver 4.0 software (Bio-Rad Laboratories, Inc.).

Phosphoprotein extracts (20 µg) from IMAC columns used for 2-DE were separated by 12% SDS-PAGE and then transferred to a nitrocellulose membrane. The western blotting procedure for phosphoproteins was conducted as previously described (18). The membrane was blocked with 5% BSA in TBS/0.05% Tween-20 (TBST) for 2 h at room temperature. After BSA saturation, the membrane was incubated overnight at 4°C with primary rabbit polyclonal antibodies against peroxiredoxin 2 (PRDX2; 1:800; cat. no. SAB2101878), protein disulfide isomerase family A member 3 (PDIA3; 1:400; cat. no. SAB2107799) and peroxiredoxin 4 (PDRX4; 1:500; cat. no. SAB4301759; all Sigma-Aldrich; Merck KGaA). The membrane was washed three times in TBST for 10 min, and then incubated for 90 min at 4°C with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (1:3,000; cat. no. G4018; Sigma-Aldrich; Merck KGaA). Protein expression was visualized by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific, Inc.), and the intensity of the signals was quantified by VersaDoc Imaging System (Bio-Rad Laboratories, Inc.). The intensities of the immunostained bands were normalized with the protein intensities measured with Red Ponceau (Bio-Rad, Laboratories, Inc.) from the same blot.

Spots from 2-DE were digested and analyzed by MS, as described by Ura et al (19). After 2-DE gel excision, the spots were washed four times with 50 mM NH₄HCO₃ and acetonitrile (ACN; Sigma-Aldrich; Merck KGaA) alternatively, and dried under vacuum in a SpeedVac system. Three µl of 12.5 ng/µl sequencing grade modified trypsin (Promega Corporation) in 50 mM NH₄HCO₃ were added to gel spots for digestion (overnight at 37°C). Peptide extraction was achieved by three changes of 50% ACN/0.1% formic acid (FA; Fluka). Peptide mixtures were dried under vacuum and dissolved in 10 µl of 5% ACN/0.1% FA and 5 µl of each sample were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) on a 6520 Q-TOF mass spectrometer (Agilent Technologies, Inc.) coupled to a chip-based chromatographic interface.

Raw data files were converted into Mascot Generic Format (MGF) files with MassHunter Qualitative Analysis Software version B.03.01 (Agilent Technologies, Inc.) and searched with Mascot Search Engine (version 2.2.4; Matrix Science) through the Proteome Discoverer Software interface (version 1.4; Thermo Fisher Scientific, Inc.). Spectra were searched against the human section of the UniProt database (version July 2018, 95,057 sequences) using the following parameters: Enzyme specificity was set to trypsin with one missed cleavage allowed, while precursor and fragment ions tolerance were set to 20 ppm and 0.05 Da, respectively. Carbamidomethyl cysteine and oxidation of methionine were set as fixed modification and variable modification, respectively. MS/MS spectra containing <5 peaks or with a total ion count <50 were discarded. Proteins were considered as positive hits if, for each protein, at least two unique peptides were identified with high confidence (FDR <0.01%). For protein spots that did not return any significant hit, a Peptide Mass Fingerprint (PMF) was also performed with Mascot. All identified proteins were verified to have phosphorylated residues in PhosphoSitePlus database (www.phosphosite.org).

Phosphorylated proteins were analyzed by PANTHER 11.0 (Protein Analysis Through Evolutionary Relationships; http://www.pantherdb.org) and Gene Ontology (http://amigo.geneontology.org/rte). Proteins were then classified according to their protein class. Since the majority of the proteins identified were involved in multiple processes, only the most relevant ones were reported.

These proteins were analyzed by Ingenuity Pathway Analysis (IPA; Qiagen GmbH), as previously described (20). Selected genes were used to generate bio-functions. For the filter summary, only high confidence associations (predicted) or that had been experimentally observed were considered.

Statistical analyses were performed with the non-parametric Wilcoxon signed-rank test for matched samples for both 2-DE and western blot data. P<0.05 was considered to indicate a statistically significant difference. All analyses were conducted with Stata/IC 14.1 for Windows (StataCorp LLC).

For phosphoproteome enrichment, an IMAC column was used. This column, although non-specific, allowed us to efficiently enrich the phosphoproteome of both the leiomyoma and the myometrium tissues. An average of 1,800 spots were detected on gels for both types of the enriched phosphoproteome. The analysis revealed 26 protein spots (Table I) with a significantly different abundance (Fig. 1) in leiomyoma tissue compared with myometrium tissues. The correlation of gel-pairs performed well, with an average matching efficiency of ~80%. In the present study, only spots corresponding to putative phosphoproteins were considered, with the following criteria: fold change in %V (where V indicates the spot volume) ≥1.5 or ≤0.6 in intensity and P<0.05. Of these spots, 25 were significantly upregulated (>1.5-fold) while one was significantly downregulated (<0.6-fold).

Western blot analysis was used to confirm the alteration of three putative phosphorylated proteins: PRDX2, PDIA3 and PDRX4. The abundance of the propsphorylated forms of these proteins in five leiomyoma samples was compared to the matched normal myometrial tissue samples (same samples as used in 2-DE analysis) by western blotting (Fig. 2). The results demonsatrted that, in the five patients tested, phosphorylation levels of PRDX2, PDIA3 and PRDX4 were markedly higher in the leiomyoma tissues compared with the myometrium tissues, consistent with the 2-DE results.

A PANTHER analysis of the identified proteins was conducted. Based on the PANTHER classification system, the results revealed that these proteins could be grouped into: Chaperone, enzyme modulator, transfer/carrier protein, cytoskeletal protein, signaling molecule and in three enzyme classes, hydrolase, ligase and oxidoreductase.

The in silico data analysis was then explanded by using the core analysis in the IPA software to construct a network in which these proteins were involved. The top networks in which these proteins were involved corresponded to: migration of cells, synthesis of ROS, cell movement, oxidative stress and cell proliferation of tumor cell lines (Fig. 3). Thirteen putative phosphoproteins were involved in the 'migration of cells' network, two were involved in the 'oxidative stress' network, five in the 'synthesis of ROS' network, 14 in the 'cell movement' network, while 13 were involved in the 'cell survival' network. Of these, four proteins [heat shock protein β1 (HSPB1), PRDX4, haptoglobin (HP) and PRDX2] suppress oxidative stress and synthesis of reactive oxygen.