Quantitative analysis of the human ovarian carcinoma mitochondrial phosphoproteome

Mitochondrial phosphorylation profiling in OCs

A total of 67 mtPPs based on 85 phosphopeptides with 124 phosphorylation sites were identified in the control and OC samples with iTRAQ-TiO₂ enrichment-LC-MS/MS between OC mitochondria relative to controls (Supplementary Table 3); and among them, 48 mitiochondrial differentially phosphorylated proteins (mtDPPs) were identified between OC and control samples. A representative MS/MS spectrum was from phosphorylated peptide ²ADELS*EK⁸ ([M + 2H]²⁺, m/z = 609.28284, S* = phosphorylated serine residue) derived from CAV1 (Swiss-Prot No.: C9JKI3) (Figure 2A), with a high-quality MS/MS spectrum, excellent signal-to-noise (S/N) ratio, and extensive product-ion b-ion and y-ion series (b₁, b₂, b₃, y₁, y₂, y₃, y₄, and y₆). The phosphorylation site was localized to amino acid residue S*₆, and the phosphorylation level was significantly decreased in OCs (T) compared to controls (N) (ratio of T/N = 0.03; p = 8.07E-07) (Supplementary Table 3). Another representative MS/MS spectrum was from phosphorylated peptide ²AS*GVAVSDGVIK¹³ ([M + 2H]²⁺, m/z = 764.89862, S* = phosphorylated serine residue) derived from CFL1 (Swiss-Prot No.: E9PS23) (Figure 2B), with a high-quality MS/MS spectrum, excellent S/N ratio, and extensive product-ion b-ion and y-ion series (b₂, b₃, b₄, b₅, b₆, y₁, y₂, y₃, y₄, y₅, y₆, y₇, and y₈). The phosphorylation site was localized to amino acid residue S*₃, and the phosphorylation level was significantly decreased in OCs compared to controls (ratio of T/N = 0.29; p = 8.25E-05) (Supplementary Table 3). The third representative MS/MS spectrum was from phosphorylated peptide ⁴¹⁹KAEDS*DS*EPEPEDNVR⁴³⁴ ([M + 3H]³⁺, m/z = 862.37878, S* = phosphorylated serine residue) derived from XRN2 (Swiss-Prot No.: B4E0B9) (Figure 2C), with a high-quality MS/MS spectrum, excellent S/N ratio, and extensive product-ion b-ion and y-ion series (b₁, b₃, b₄, b₅, b₆, b₇, b₈, b₁₀, y₁, y₃, y₆, y₈, y₉, and y₁₀). The phosphorylation site was localized to amino acid residues S*₄₂₃ and S*₄₂₅, and the phosphorylation level was not significantly changed in OCs compared to controls (ratio of T/N = 0.86, p = 9.16E-02) (Supplementary Table 3). With the same method, each phosphorylated peptide and phosphorylation site was identified with MS/MS data, and quantified (Supplementary Table 3). Among 67 mtPPs, 18 identified proteins were reported by previous studies in OCs [17], including ANK1, ATP2A2, BNIP3L, CAV1, CFL1, CTNNA1, DSTN, EHD2, FTMT, HDGF, HMGA1, HSP90AA1, HSPD1, MARCKS, OSBP, PGRMC1, PSIP1, and TMEM45A (Figure 1). In terms of mechanism, only CFL1 showed that phosphorylated CFL1 was associated with taxol chemotherapy resistance in human OC cells [18]. The other mtPPs were never reported before. Most of identified phosphorylated sites were occurred at residue Ser, and a few phosphorylated sites were at residues Thr and Tyr. Some mtPPs were phosphorylated at more than one Ser-residues, including BNIP3L, BZW1, DKFZp686M0430, EIF2B5, HMGA1, MYH10, aging-associated protein 14a, OSBP, PDAP1, PKP2, PRKAR2A, PTDSS1, RAB28, RPLP0, STT3B, RPLP2, TOMM20, and XRN2. Some mtPPs were phosphorylated at both residues Ser and Thr, such as PSIP1 (pS₂₇₁, pT₂₇₂, pS₂₇₃, and pS₂₇₅), and PUS1 (pS₁₂₇, and pT₁₃₃). Some mtPPs were phosphorylated at residue Thr, such as KIN27 (pT₁₇₉, and pT₁₈₁). Some mtPPs were phosphorylated at both residues Ser and Tyr, such as NBAS (pS₄₇₃, pS₄₇₅, and pY₄₇₇).

GO enrichment analysis of mtPPs

Those 67 mtPPs between OCs and controls were analyzed with GO enrichment methods, in biological process (BP) (Figure 3A, and Supplementary Table 1), cellular component (CC) (Figure 3B, and Supplementary Table 1), and molecular function (MF) (Figure 3C, and Supplementary Table 1). BP analysis revealed five important biological processes to involve in mtPPs, including anion transmembrane transport, protein targeting to mitochondrion, gap junction assembly, regulation of autophagy of mitochondrion, and actin filament fragmentation. The proteins enriched in the same cluster had biological similarity. CC analysis revealed that mtPPs were mainly involved in cytosolic large ribosomal subunit, eukaryotic translation initiation factor 2B complex, eukaryotic translation initiation factor 4F complex, eukaryotic translation initiation factor 2B complex, proteasome core complex, alpha-subunit complex, germinal vesicle, cAMP-dependent protein kinase regulator activity, U2AF, mitochondrial outer membrane, actin filament bundle, outer mitochondrial membrane protein complex, smooth endoplasmic reticulum part, cyclin-dependent protein kinase activating kinase holoenzyme complex, plasma membrane raft, heterochromatin, mitochondrial alpha-ketoglutarate dehydrogenase complex, and Dsl1/NZR complex. Moreover, some phosphoproteins that were annotated within other cellular compartments but not within mitochondria were also detected in this study, which might be due to interaction with the proteins that are situated at the outer mitochondrial membrane or mitochondria-related proteins. MF analysis revealed that mtPPs were mainly distributed in protein kinase C binding, vinculin binding, actin filament binding, ADP binding, cAMP-dependent protein kinase regulator activity, carbon-oxygen lyase activity, porin activity, translation initiation factor activity, ferroxidase activity, sulfide:quinone oxidoreductase activity, protein channel activity, apolipoprotein A-I binding, transcription termination site sequence-specific DNA binding, supercoiled DNA binding, inward rectifier potassium channel inhibitor activity, alpha-ketoacid dehydrogenase activity, dol-P-Man: Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase activity, pre-mRNA 3'-splice site binding, and lutropin-choriogonadotropic hormone receptor binding. Some mtPPs also played roles in multiple biological functions.

PPI network of mtPPs

The PPI network of 67 mtPPs was constructed with String analysis database (Figure 4A, and Supplementary Table 2). First, the PPI results revealed 7 mtPP-mtPP pairs had high combined score (value > 0.9) and good correlations (value > 0.4), including RPLP0 and EIF2S2, RPLP0 and MRPS16, DAC2 and VDAC1, RPLP0 and PA2G4, PTGES3 and HSP90AA1, HSPD1 and HSP90AA1, and RPLP0 and RPLP2. Those matched mtPP-mtPP pairs might exist direct or indirect binding sites and co-expression relationship. Second, the PPI results also revealed 20 mtPP-mtPP pairs had high combined score (value>0.9) but low correlations (value>0.4), including CAV1 and HSP90AA1, HSP90AA1 and PRKACA, HMGA1 and HIST1H1E, HMGA1 and HIST1H1C, EIF4G3 and EIF2S2, CFL1 and HSP90AA1, VDAC3 and VDAC2, HSPD1 and TOMM22, MYH10 and MYH9, EIF2S2 and EIF2B5, VDAC3 and VDAC1, VDAC2 and TOMM20, TOMM20 and VDAC1, VDAC3 and TOMM20, HSPD1 and TOMM20, HSPD1 and VDAC1, TOMM20 and TOMM22, EIF2S2 and RPLP2, HIST1H1C and HIST1H1E, and PRKACA and PRKAR2A. The mutual influence between two mtPPs might be though protein-protein binding but not regulating expressions.

Significant signaling pathways involved in mtPPs

CooLGeN (Version GenCLiP 3) was used to map 67 mtPPs to the corresponding molecules (genes; proteins) for pathway network analysis. A total of 32 statistically significantly signaling pathway systems (p < 0.05) were identified with 67 mtPPs, and were grouped into 15 clusters (Figure 4B, and Table 1). Cluster 1 included mitophagy, and mitochondrial protein import pathways. Cluster 2 included apoptosis pathway. Cluster 3 included Ub-specific processing proteases, and deubiquitination pathways. Cluster 4 included vascular endothelial growth factor (VEGF), and TGF beta receptor signaling. Cluster 5 included RHO GTPases-activated ROCKs, EPH-Ephrin signaling, semaphoring interactions, regulation of actin cytoskeleton, and RHO GTPase effectors. Cluster 6 included VEGFA VEGFR2 signaling pathway. Cluster 7 included mitochondrial calcium ion transport. Cluster 8 included eukaryotic translation initiation, cap dependent translation initiation, translation, and ribosome. Cluster 9 included necroptosis, and NOD-like receptor signaling pathway. Cluster 10 included translation factors, and RNA transport. Cluster 11 included cGMP-PKG signaling pathway, calcium signaling pathway, and transport of small molecules. Cluster 12 included metabolism of proteins. Cluster 13 included the validated targets of C-MYC transcriptional activation, cellular responses to external stimuli, and cellular responses to stress. Cluster 14 included Fc gamma R-mediated phagocytosis. Cluster 15 included L13a-mediated translational silencing of ceruloplasmin expression, and GTP hydrolysis and joining of the 60S ribosomal subunit.

When mitochondria were in the status of imbalance, such as stress or damage, defective mitochondria might enter selective degradation process by autophagy, named mitophagy. Mitophagy is a crucial mechanism of quality control in mitochondria and can be regarded as a double-edged sword in cancer cells. The defective mitochondria that responded to oncogenic stresses could either stimulate or block tumorigenesis through mitophagy [19]. Here, the new biomarkers that were found in the mitophagy pathway included phosphorylations of TOMM20 at residues S₁₃₅ and S₁₃₈ (fold change = 3.54, p = 1.00E-04), TOMM20 at residue S₁₃₅ (fold change = 2.56, p = 8.00E-05), TOMM22 at residue S₁₅ (fold change = 1.16, p = 1.00E-05), and VDAC1 at residue S₁₀₄ (fold change = 2.63, p = 1.00E-04), which might be novel therapeutic targets and fundamental improvement of the damaged mitochondria in OC patients. These data showed that mitophagy might play important role in mediating tumorigenesis and tumor progression.

VEGF signaling is related to many physiological and pathological processes in human. VEGF is a specific heparin-binding growth factor of vascular endothelial cells, which can induce angiogenesis in vivo. At the same time, VEGF can increase vascular permeability and mobility of cancer cells, induce tumor angiogenesis, and maintain the growth of tumors [20]. Here, the new biomarkers that were found in the VEGF signaling included phosphorylations of CAV1 at residue S₆ (fold change = 0.03, p = 8.07E-07), CAV1 at residue S₂₆ (fold change = 0.06, p = 9.35E-09), CTNNA1 at residue S₅₁₈ (fold change = 0.78, p = 1.00E-04), HSP90 at residue S₂₁₇ (fold change = 0.80, p =2.83E-02), CFL1 at residue S₃ (fold change = 0.29, p = 8.00E-05), and MYH9 at residue S₁₉₄₃ (fold change = 1.40, p = 7.70E-03), which might be novel molecules and regulation mechanism for tumor angiogenesis.

Rho GTPases are the members of small GTPases, and Rho GTPase pathway is closely related to cytoskeleton reorganization, cell morphology, apoptosis, cell migration, and cell cycle. Recent studies showed that Rho GTPases mediated tumor invasion and metastasis by regulating extracellular matrix remodeling, loss of epithelial polarity, lymphatic vasculature, cytoskeleton reorganization, and between-cell junction and adhesion [21]. Here, the new biomarkers that were found in Rho GTPase signaling included phosphorylations of CFL1 at residue S₃ (fold change = 0.29, p = 8.00E-05), MYH10 at residue S₁₉₇₅ (fold change = 0.82, p = 3.80E-01), MYH10 at residue S₁₉₅₆ (fold change = 0.80, p = 3.68E-01), MYH10 at residues S₁₉₇₅ and S₁₉₅₆ (fold change = 1.77, p = 3.99E-02), MYH9 at residue S₁₉₄₃ (fold change = 1.40, p = 7.70E-03), and CTNNA1 at residue S₅₁₈ (fold change = 0.78, p = 1.00E-04), which might be novel molecules and regulation mechanism for tumor invasion and metastasis.

Multiple signal transduction pathways are initiated by calcium ions (Ca²⁺), so intracellular calcium ions are the necessary condition for occurrence, development, and proliferation of tumors through various pathways [22]. Mitochondrial calcium ion transport pathway is important in regulating Ca²⁺ concentrations in cells. This study found that mitochondrial calcium ion transport pathway and calcium signaling pathway were enriched to involve mtPPs, including phosphorylations of VDAC1 at residue S₁₀₄ (fold change = 2.63, p = 1.00E-04), VDAC2 at residue S₁₄₀ (fold change = 1.99, p = 7.90E-03), VDAC3 at residue T₆, and ATP2A2 at residue S₅₅₄ (fold change = 0.69, p = 2.39E-02). Imbalance of Ca²⁺ might be the cause of tumorigenesis, so it is definitely worth further exploring.

C-Myc-mediated transcriptional activity promotes tumorigenesis and development. The major oncogenic mechanisms of c-MYC are related to promotion of cell cycle, drug resistance, reprogramming of cell metabolism, and rapid proliferation [23]. Here, the new biomarkers that were found in C-MYC-mediated transcriptional activation pathway included phosphorylations of HMGA1 at residues S₉₂ and S₉₃ (fold change = 2.02, p = 1.70E-03), and at residues S₈₈, S₉₂, and S₉₃, HSP90 at residue S₂₁₇ (fold change = 0.80, p = 2.83E-02), and HSPD1 at residue S₇₀ (fold change = 2.34, p = 3.40E-03). Enhanced c-Myc-mediated transcriptional activity might induce cancer.

Relationships of phosphorylated sites and functional domains in mtPPs

A total of 29 identified mtPPs contained a certain structural and functional domains (Figure 5). Conjoint analysis of protein domains of mtPPs provided novel insights into mitochondrial protein phosphorylation and its potential role in OCs. First, this study found that ANK1, ANKIB1, EIF2B5, HMGA1, CS, PSMA3, and VANGL2 had phosphorylation sites within protein domains. ANK1 contained ankyrin-repeated region circular (sites: 11-786), with phosphorylation at residue S₈₉. ANKIB1 also contained ankyrin-repeated region circular (sites: 45-197), with phosphorylation at residue S₈₉. EIF2B5 contained W2 domain (sites: 543-720), with phosphorylation at residues S₇₁₇ and S₇₁₈. HMGA1 contained HMGI_Y domain (sites: 21-89), with phosphorylation at residue S₈₈. PSMA3 contained proteasome alpha domain (sites: 8-30 and 23-237), with phosphorylation at residue S₇₈. VANGL2 contained TPR regions (sites: 114-578), with phosphorylation at S₅₁₇. Second, CFL1 and HMGA1 were phosphorylated near protein domains. CFL1 contained actin-depolymerising factor homology domain (ADF-H domain) (sites: 4-153), with phosphorylation at residue S₃. HMGA1 contained HMG-Y DNA-binding domain (AT-hooks) (sites: 21-89), with phosphorylation at residues S₉₂ and S₉₃. Third, the phosphorylation sites of the other 20 mtPPs with structural and functional domains were neither presented within domains nor near domains. However, it doesn't mean that those mtPPs were not important. The between-molecule abscopal effects could not been ignored. Additionally, phosphorylation at residue Y was a low abundance event compared to that at residues S and T. Specific tyrosine kinase inhibitors had been discovered to have some pretty amazing therapeutic effects in different types of cancers. NBAS contained ribosomal protein S14 signature (sites: 2207-2229), with phosphorylations at residues S₄₇₃, S₄₇₅ and Y₄₇₇. This protein was associated with transporting proteins from Golgi complex to endoplasmic reticulum.

Upstream analysis indicated potential regulatory mechanism

Upstream regulation analysis of 67 mtPPs indicated potential regulatory mechanisms and provided potential anti-cancer drugs (Figure 6 and Table 2). Upstream regulation analysis showed that TP53 might be an inhibitor for PRKAR2A, MYH9, MYH10, HSPD1, EIF4G3, ANK1, HSP90AB1, HMGA1, and PA2G4. In human, transforming growth factor beta 1 (TGF-β1) was encoded by TGFB1 gene, which was a secreted protein. Upstream regulation analysis showed that TGFB1 might be an inhibitor for PGRMC1, HMGA1, VDAC2, MYH9, OSTF1, HDGF, BNIP3L, PA2G4, CFL1, and SNTB2. Dexamethasone was a kind of corticosteroid medication. Upstream regulation analysis showed that dexamethasone might be an inhibitor for HMGA1, OSTF1, ATP2A2, BCKDHA, MARCKS, EIF4G3, CFL1, and RPLP0. Thapsigargin, as a tumor promoter in mammalian cells, was non-competitive inhibitor of sarcoplasmic and endoplasmic reticulum Ca²⁺ ATPase. Upstream regulation analysis showed that dexamethasone might be an inhibitor for ATP2A2, and CAV1. L-dopa was also known as L-3,4-dihydroxyphenylalanine. Upstream regulation analysis showed that L-dopa might be an activator for VDAC1, PKP2, CLN6, VANGL2, ATP2A2, and PA2G4. Forskolin was a commonly used chemical toxicant in laboratory to increase cyclic AMP level. Upstream regulation analysis showed that forskolin might be an activator for PGRMC1, OSTF1, BNIP3L, and PRKAR2A.

Cancer and control tissues

Frozen OC tissues (n = 8) and control ovaries with benign gynecologic disease (n = 11) were obtained from Department of Gynecology, Xiangya Hospital, Central South University, China. This study was approved by the Medical Ethics Committee of Xiangya Hospital, and the written informed consent was obtained from each patient. Both OC and control tissues were verified with histological analysis. Each tissue sample was immediately placed in liquid nitrogen and then stored at −80°C.

Preparation of mitochondrial proteins

Ovarian tissue samples were fully minced in pieces and homogenized in mitochondrial isolation buffer that contained Nagarse, followed by differential-speed centrifugation to obtain crude mitochondria. The crude mitochondria were further purified with Nycodenz-gradient centrifugation. The purified mitochondria samples were verified by western blot and electron microscopy. The detailed experimental procedure was described previously [4, 5]. The prepared OC and control mitochondria samples were used to extracted proteins with SDT lysis buffer that contained 4% SDS, 1 mM DTT, and 100 mM Tris-HCl pH 7.6, followed by centrifugation (14,000 g, 40 min) to collect the supernatant as extracted mitochondrial proteins. The protein content was quantified with the BCA Protein Assay Kit (Bio-Rad, USA).

iTRAQ labeling

An amount (200 μg) of extracted mitochondrial proteins were mixed with 30 μl solution that contained 4% SDS, 100 mM DTT, and 150 mM Tris-HCl pH 8.0. The detergent, DTT, and other low-molecular-weight components were removed by repeated ultrafiltration with the solution of 8 M urea and 150 mM Tris-HCl pH 8.0. The reduced cysteine residues were blocked with adding 100 μl solution that contained 100 mM iodoacetamide, 8 M urea, and 150 mM Tris-HCl pH 8.0, for incubation in darkness for 30 min, followed by washing with 100 μl solution (8 M urea and 150 mM Tris-HCl pH 8.0) for 3 times and then 100 μl dissolution buffer for 2 times. The protein suspensions were digested with 4 μg trypsin (Promega) (37 °C, overnight). The tryptic peptides were desalted on C18 Cartridges, concentrated by vacuum centrifugation. The peptide content was estimated by UV light spectral density at 280 nm. A amount (100 μg) of tryptic peptide mixture of each sample was labeled using iTRAQ reagent. Each sample was labeled by three different iTRAQ reagents. The detailed experimental procedure was described previously [4, 5].

Enrichment of phosphopeptides

Six iTRAQ-labeled peptides were equally mixed, concentrated by a vacuum concentrator, and resuspended in 500 μl DHB buffer. TiO₂ beads were added and agitated for 2 h, followed by centrifugation (1 min, 5000 g) to keep the beads, washing with 50 μL of washing buffer I (30% acetonitrile and 3% trifluoroacetic acid) (3 x) and then 50 μL of washing buffer II (80% acetonitrile and 0.3% trifuoroacetic acid) (3 x) to remove the remaining non-adsorbed material. Finally, phosphopeptides were eluted with 50 μL of elution buffer (40% acetonitrile and 15% NH₃·H₂O) (3 x), and then lyophilized.

LC-MS/MS of enriched phosphopeptides

The TiO₂-enriched phosphopeptides were analyzed with LC-MS/MS. Briefly, the enriched phosphopeptides were loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100 μm x 2 cm, nanoViper C18) connected to the C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin) in buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl/min controlled by IntelliFlow technology. The linear gradient was 0–55% buffer B for 220 min, 55-100% buffer B for 8 min, and then 100% buffer B for 12 min. MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific), with the following parameters, including positive ion mode, data-dependent top10 method to choose the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation, automatic gain control (AGC) target 3e6, and maximum inject time to 10 ms, dynamic exclusion duration 40.0 s, resolution for MS set to 70,000 at m/z 200, resolution for MS/MS set to 17,500 at m/z 200, isolation width set to 2 m/z, normalized collision energy set to 30 eV, the underfill ratio set to 0.1%. MS/MS spectra were used to search protein database with MASCOT engine (Matrix Science, London, UK; version 2.2) embedded into Proteome Discoverer 1.4. The intensities of iTRAQ reporter ions were used to determine differentially phosphorylated proteins between OC and control mitochondrial samples.

Bioinformatics analysis

The reported biomarkers for OC based on the identified mtPPs were obtained from NCBI (https://www.ncbi.nlm.nih.gov/pubmed/). Gene ontology, including BP and CC, was analyzed with Cytoscape ClueGO to obtain more insights into the biological information of mtPPs. PPI network was analyzed with String database (http://string-db.org/cgi/input.pl). Pathway enrichment was analyzed by CooLGeN (http://ci.smu.edu.cn/CooLGeN/Home.php) based on Reactome, Wikipathways, NetPath, and KEGG database. Each MS/MS-derived phosphoprotein amino acid sequence was input into the ScanProsite program (http://prosite.expasy.org/scanprosite) to determine its protein domains and relationship of phosphorylation sites and protein domains. The Swiss-Prot accession numbers and corresponding fold-changes between OCs and controls were input to the Ingenuity Pathway Analysis (IPA) data-upload workflow. The upstream regulation analysis was generated to involve those phosphoproteins.

Statistical analysis

The Student’s t-test was used to assess mtDPPs between OC and control groups. P-value for GO enrichment analysis was obtained by two-sided hypergeometric test and corrected by Benjamini-Hochberg. P-value for pathway enrichment analysis was obtained by two-sided hypergeometric test and corrected by Q-value. The level of statistical significance was set as p < 0.05.

Ethical approval

All the patients were informed about the purposes of the study and consequently have signed their “consent of the patient”. All investigations conformed to the principles outlined in the Declaration of Helsinki and were performed with permission by the responsible Medical Ethics Committee of Xiangya Hospital, Central South University, China.