IJC International Journal of Cancer
V-ATPase inhibition by archazolid leads to lysosomal dysfunction resulting in impaired cathepsin B activation in vivo €hlich2, Georg J. Arnold2, Laura Schreiner1, Karin von Schwarzenberg3, Andreas Roidl1, Rebekka Kubisch1, Thomas Fro 3 Angelika M. Vollmar and Ernst Wagner1 1
Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig Maximilians University, Munich, Germany Laboratory for Functional Genome Analysis, Gene Center of the Ludwig Maximilians University, Munich, Germany 3 Pharmaceutical Biology, Department of Pharmacy, Ludwig Maximilians University, Munich, Germany 2
The myxobacterial agent archazolid inhibits the vacuolar proton pump V-ATPase. V-ATPases are ubiquitously expressed ATPdependent proton pumps, which are known to regulate the pH in endomembrane systems and thus play a crucial role in endo- and exocytotic processes of the cell. As cancer cells depend on a highly active secretion of proteolytic proteins in order to invade tissue and form metastases, inhibition of V-ATPase is proposed to affect the secretion profile of cancer cells and thus potentially abrogate their metastatic properties. Archazolid is a novel V-ATPase inhibitor. Here, we show that the secretion pattern of archazolid treated cancer cells includes various prometastatic lysosomal proteins like cathepsin A, B, C, D and Z. In particular, archazolid induced the secretion of the proforms of cathepsin B and D. Archazolid treatment abrogates the cathepsin B maturation process leading to reduced intracellular mature cathepsin B protein abundance and finally decreased cathepsin B activity, by inhibiting mannose-6-phoshate receptor-dependent trafficking. Importantly, in vivo reduced cathepsin B protein as well as a decreased proteolytic cathepsin B activity was detected in tumor tissue of archazolid-treated mice. Our results show that inhibition of V-ATPase by archazolid reduces the activity of prometastatic proteases like cathepsin B in vitro and in vivo.
Cancer Therapy
Vacuolar (H1)-ATPases (V-ATPases) are found on various membranes, including lysosomes, endosomes, vesicles and the plasma membrane. As V-ATPases are ATP-dependent proton pumps they are crucial for maintaining the pH of these compartKey words: archazolid, cancer, cathepsin B, metastasis, V-ATPase Abbreviations: ER: endoplasmic reticulum; LC-MS/MS: liquid chromatography–tandem mass spectrometry; M6PR: mannose-6phosphate; mCTSB: mature cathepsin B; PLC: prelysosomal compartment; proCTSB: procathepsin B; TGN: trans-Golgi network; V0c: V-ATPase domain V0 subunit c Additional Supporting Information may be found in the online version of this article. Grant sponsor: German Research Foundation (DFG); Grant numbers: FOR 1406, Vo 376-14/15 and WA1648/3-1 DOI: 10.1002/ijc.28562 History: Received 12 July 2013; Accepted 8 Oct 2013; Online 25 Oct 2013 Correspondence to: Rebekka Kubisch, Pharmaceutical Biotechnology, Department of Pharmacy, Center for System-based Drug Research, Ludwig-Maximilians University, Butenandtstr. 5-13, 81377 Munich, Germany, Tel.: 149-89-2180-77840, Fax: 149-89-2180-77791, E-mail: [email protected]; or Ernst Wagner, Pharmaceutical Biotechnology, Department of Pharmacy, Center for System-based Drug Research, LudwigMaximilians University, Butenandtstr. 5-13, 81377 Munich, Germany, Tel.: 149-89-2180-77840, Fax: 149-89-2180-77791, E-mail: [email protected]
C 2013 UICC Int. J. Cancer: 134, 2478–2488 (2014) V
ments. Thus, they play a vital role in various cellular processes, like intracellular targeting of lysosomal enzymes, protein processing and degradation as well as receptor-mediated endocytosis.1 In cancer, a combination of increased metabolism and reduced oxygen and nutrient supply leads to an acidic tumor environment.2 Therefore, tumors have to strictly control their intracellular pH. V-ATPases are reported to play an important role in tumor progression by controlling the pH-homeostasis.3–5 Therefore, compounds inhibiting V-ATPase are proposed as novel drugs for cancer therapy.4 The plecomacrolides bafilomycin and concanamycin as well as archazolid are natural compounds,6–9 which efficiently inhibit V-ATPase by binding to the V0c subunit.10–14 The archazolids are composed of a macrocyclic lactone ring with a thiazole side chain and were first isolated by Sasse et al. from cultivated myxobacteria Archangium gephyra.6 Moreover, archazolid specifically inhibits the V-ATPase in nanomolar range compared to purified Na1/K1-ATPases and mitochondrial F-ATPase.11 Archazolid was very recently shown to inhibit migration of cancer cells in vitro and to reduce the lung colonialization of murine breast cancer cells in vivo.15 During metastasis tumor cells are invading the surrounding tissue and migrate to distant sites. One of the crucial steps in this process is the degradation of extracellular matrix by extracellular proteases.16–18 Some of these prometastatic proteases are members of the cathepsin family. However, their role in the metastatic process is discussed controversially. Some authors state that extracellular cathepsins including
2479
Kubisch et al.
What’s new? Many tumor cells secrete proteases such as cathepsins, which are thought to enhance their ability to invade tissues and metastasize. V-ATPase inhibitors are known to inhibit metastases in mice. In this study, the authors tie these two lines of study together: They found that the V-ATPase inhibitor archazolid impairs the secretion of mature proteases such as cathepsin B and D, causing the proenzyme forms to be released instead. This represents a novel mechanism that may further explain the therapeutic effects of a promising new class of chemotherapeutic agents.
Material and Methods Compounds
Bafilomycin A1, concanamycin A, tunicamycin, brefeldin, NH4Cl, doxorubicin and chloroquine were purchased from Sigma Aldrich, archazolid A was purified and isolated as described previously,6 archazolid B was chemically synthesized by Roethle et al.36 Both compounds were dissolved in dimethyl sulfoxide (DMSO) and show similar bioactivity.15 For all in vitro experiments, archazolid B was used. For all in vivo experiments, archazolid A was used. Cell culture
T-24 (DSMZ ACC 376, provided by Barbara Mayer, Department of Surgery, Clinic Grosshadern, University of Munich, C 2013 UICC Int. J. Cancer: 134, 2478–2488 (2014) V
Germany) cells were cultured in McCoy’s modified 5A media (Biochrom, Germany) supplemented with 10% FCS Biochrom, Germany. 4T1luc cells (Caliper life science) were cultured in RPMI 1640 media (Invitrogen) supplemented with 10% FCS. MCF-7 cells (Cell line service, Germany) were cultured in DMEM 4.5 g/L glucose (Invitrogen) supplemented with 20% FCS. Cells were grown at 37 C, 5% CO2 in a humidified atmosphere. Cell viability assay (CellTiterGlo)
To analyze the cell viability of T-24 cells the CellTiterGlo (Promega, Germany) assay was applied. This assay detects the ATP content of a given sample, thus indicating the relative amount of metabolizing, living cells in an ATP-dependent luciferase reaction. The assay was performed according to the manufacturer’s instructions. Briefly, 5 3 104 cells per well were seeded in 96-well plates. Twenty-four hours after seeding cells were treated in FCS free media. After compound incubation, half of the volume of each well was replaced by the reaction buffer. The luminescence was recorded using a Luminometer Lumat LB9507 instrument (Berthold, Germany). Results were calculated as percent of control. Collection of conditioned media
1 3 106 MCF-7 or T24 cells were seeded in 10 cm dishes (Nunc). Twenty-four hours after seeding cells were washed three times (for proteomic analysis) or one time (western blot) with FCS-free media. After treatment of the cells in 10 mL of FCS-free media, supernatants were harvested, followed by centrifugation of 5 min at 1,000 rpm to remove cells and cell debris. Conditioned media samples were concentrated using Amicon Ultra-4 Centrifugal Filter Units (Merck, Millipore); cutoff 3 kDa for proteomics and 10 kDa for western blot. SDS-PAGE
SDS-PAGE was performed according to Laemmli et al.37 Conditioned media (30 mL) or 15–50 mg of total protein lysate were supplemented with Laemmli buffer and boiled for 5 min at 95 C for protein denaturation. Samples were loaded on 10 or 12.5% SDS-PA-gels. Protein separation was performed at 0.03 mA per gel for approximately 2 hr. In-gel-digestion
After electrophoresis, each lane was cut into 10 slices. Each piece was transferred into a reaction tube and covered with 45 mM DTT (Sigma Aldrich) in 50 mM NH4HCO3 (Sigma
Cancer Therapy
cathepsin B directly contribute to the matrix degradation.19–21 In contrast, others show that intracellular or membrane bound cathepsin B is crucial for extracellular matrix degradation by other proteases like urokinase (uPA) or matrix metalloproteinases.22,23 Nevertheless, cathepsin B expression is increased in different types of cancer like prostate cancer, oesophageal adenocarcinoma, breast cancer or colorectal cancer.24–30 Cathepsin B (CTSB) is synthesized at the rough endoplasmic reticulum as preprocathepsin B. Signal peptides target preproCTSB to the ER lumen, where it becomes cleaved to proCTSB. Subsequently, procathepsin B is transported through the ER to the Golgi apparatus. In the trans-Golgi network (TGN), it is bound after glycosylation and phosphorylation to membrane-associated mannose-6-phosphate receptor (M6PR) and proCTSB bound to M6PR containing vesicles are formed. These vesicles are directed to prelysosomal compartments (PLCs). During the lysosome maturation process, the pH of these PLC is decreasing which leads to the release of proCTSB from the M6PR and to the recycling of the receptor to the TGN. PLC are fusing with lysosomes and proCTSB is subsequently cleaved to active CTSB. Two forms of active CTSB are known: (i) the one chain form (31 kDA) and (ii) the two chain form (25/26 1 5 kDA).31–33 As extracellular signaling and cell–cell communications play an important role during metastatic processes34 and VATPase inhibitors modify endo- and exocytotic events by altering membrane trafficking15,35 the following hypothesis arouse: archazolid alters the secretion profile of invasive tumor cells contributing to its antimetastatic potential. We analyzed the secretome of archazolid-treated migratory carcinoma cells, found an impaired cathepsin B maturation process and evaluated its functional impact in vitro as well as in vivo.
2480
V-ATPase inhibition leads to impaired CTSB activation
Aldrich). After an incubation of 30 min at 55 C gel-pieces were covered with 100 mM iodoacteamide (Sigma Aldrich) in 50 mM NH4HCO3 for 15 min at RT in the dark. This step was repeated. Subsequently, gel-pieces were washed two time using 50 mM NH4HCO3. For tryptic digestion, pieces were chopped using a pipet tip and covered with 50 mM NH4HCO3 containing 70 ng trypsin (Promega, Germany) to 50 mg of protein. Incubation was carried out at 37 C overnight. After the digestion, supernatants were collected and pieces covered with 70% acetonitrile for at least 10 min. Supernatants were combined afterwards. After freeze-drying, peptide samples were stored at 280 C until LC-MS/MS measurement.
Cancer Therapy
LC-MS/MS analysis
Nano-LC separation was done with a nanoliquid chromatography system (Ettan MDLC, GE Healthcare). Peptide samples were loaded on a trap column (10 mL/min, loading buffer: 0.1% formic acid; trap column: C18 PepMap 100, 5-mm bead size, 300 mm i.d., 5 mm length, LC Packings) and separated with an analytical reversed phase column (Reprosil-Pur C18 AQ, 3 mm; 150 mm 3 75 mm, Dr Maisch, Germany) using a 30-min gradient from 0% B to 60% B (solvent A: 0.1% formic acid; solvent B: 84% CH3CN/0.1% formic acid), at a flow rate of 280 nL/min. For electrospray ionization, a distal coated SilicaTips (FS-360-20-10-D-20, New Objective) and a needle voltage of 1.7 kV was used. Tandem mass spectrometry was performed with an Orbitrap XL mass spectrometer (Thermo Scientific). MS and MS/MS spectra were acquired using cycles of one MS scan (mass range m/z 300–2000) and five subsequent data-dependent CID MS/MS scans (“dynamic exclusionTM activated”; collision energy: 35%). RAW data were processed using MASCOT Daemon and MASCOT Server (V2.3, Matrix Science, Boston) with the human subset of the SwissProt Database (Release 2012_11) and the following parameters: (i) “Fixed modifications”: Carbamidomethyl (C); (ii) Variable modifications: Oxidation (M); (iii) Decoy database: checked; (iv) Peptide charge: 11, 21 and 31; (v) Peptide tol. 6: 2 Da; (vi) MS/MS tol. 6: 0.8 Da. MASCOT data files were further evaluated using Scaffold 2.04 (Proteome Software), filtered identifications with at least two individual peptides (>95%) and a total false discovery rate
V-ATPase inhibition by archazolid leads to lysosomal dysfunction resulting in impaired cathepsin B activation in vivo €hlich2, Georg J. Arnold2, Laura Schreiner1, Karin von Schwarzenberg3, Andreas Roidl1, Rebekka Kubisch1, Thomas Fro 3 Angelika M. Vollmar and Ernst Wagner1 1
Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig Maximilians University, Munich, Germany Laboratory for Functional Genome Analysis, Gene Center of the Ludwig Maximilians University, Munich, Germany 3 Pharmaceutical Biology, Department of Pharmacy, Ludwig Maximilians University, Munich, Germany 2
The myxobacterial agent archazolid inhibits the vacuolar proton pump V-ATPase. V-ATPases are ubiquitously expressed ATPdependent proton pumps, which are known to regulate the pH in endomembrane systems and thus play a crucial role in endo- and exocytotic processes of the cell. As cancer cells depend on a highly active secretion of proteolytic proteins in order to invade tissue and form metastases, inhibition of V-ATPase is proposed to affect the secretion profile of cancer cells and thus potentially abrogate their metastatic properties. Archazolid is a novel V-ATPase inhibitor. Here, we show that the secretion pattern of archazolid treated cancer cells includes various prometastatic lysosomal proteins like cathepsin A, B, C, D and Z. In particular, archazolid induced the secretion of the proforms of cathepsin B and D. Archazolid treatment abrogates the cathepsin B maturation process leading to reduced intracellular mature cathepsin B protein abundance and finally decreased cathepsin B activity, by inhibiting mannose-6-phoshate receptor-dependent trafficking. Importantly, in vivo reduced cathepsin B protein as well as a decreased proteolytic cathepsin B activity was detected in tumor tissue of archazolid-treated mice. Our results show that inhibition of V-ATPase by archazolid reduces the activity of prometastatic proteases like cathepsin B in vitro and in vivo.
Cancer Therapy
Vacuolar (H1)-ATPases (V-ATPases) are found on various membranes, including lysosomes, endosomes, vesicles and the plasma membrane. As V-ATPases are ATP-dependent proton pumps they are crucial for maintaining the pH of these compartKey words: archazolid, cancer, cathepsin B, metastasis, V-ATPase Abbreviations: ER: endoplasmic reticulum; LC-MS/MS: liquid chromatography–tandem mass spectrometry; M6PR: mannose-6phosphate; mCTSB: mature cathepsin B; PLC: prelysosomal compartment; proCTSB: procathepsin B; TGN: trans-Golgi network; V0c: V-ATPase domain V0 subunit c Additional Supporting Information may be found in the online version of this article. Grant sponsor: German Research Foundation (DFG); Grant numbers: FOR 1406, Vo 376-14/15 and WA1648/3-1 DOI: 10.1002/ijc.28562 History: Received 12 July 2013; Accepted 8 Oct 2013; Online 25 Oct 2013 Correspondence to: Rebekka Kubisch, Pharmaceutical Biotechnology, Department of Pharmacy, Center for System-based Drug Research, Ludwig-Maximilians University, Butenandtstr. 5-13, 81377 Munich, Germany, Tel.: 149-89-2180-77840, Fax: 149-89-2180-77791, E-mail: [email protected]; or Ernst Wagner, Pharmaceutical Biotechnology, Department of Pharmacy, Center for System-based Drug Research, LudwigMaximilians University, Butenandtstr. 5-13, 81377 Munich, Germany, Tel.: 149-89-2180-77840, Fax: 149-89-2180-77791, E-mail: [email protected]
C 2013 UICC Int. J. Cancer: 134, 2478–2488 (2014) V
ments. Thus, they play a vital role in various cellular processes, like intracellular targeting of lysosomal enzymes, protein processing and degradation as well as receptor-mediated endocytosis.1 In cancer, a combination of increased metabolism and reduced oxygen and nutrient supply leads to an acidic tumor environment.2 Therefore, tumors have to strictly control their intracellular pH. V-ATPases are reported to play an important role in tumor progression by controlling the pH-homeostasis.3–5 Therefore, compounds inhibiting V-ATPase are proposed as novel drugs for cancer therapy.4 The plecomacrolides bafilomycin and concanamycin as well as archazolid are natural compounds,6–9 which efficiently inhibit V-ATPase by binding to the V0c subunit.10–14 The archazolids are composed of a macrocyclic lactone ring with a thiazole side chain and were first isolated by Sasse et al. from cultivated myxobacteria Archangium gephyra.6 Moreover, archazolid specifically inhibits the V-ATPase in nanomolar range compared to purified Na1/K1-ATPases and mitochondrial F-ATPase.11 Archazolid was very recently shown to inhibit migration of cancer cells in vitro and to reduce the lung colonialization of murine breast cancer cells in vivo.15 During metastasis tumor cells are invading the surrounding tissue and migrate to distant sites. One of the crucial steps in this process is the degradation of extracellular matrix by extracellular proteases.16–18 Some of these prometastatic proteases are members of the cathepsin family. However, their role in the metastatic process is discussed controversially. Some authors state that extracellular cathepsins including
2479
Kubisch et al.
What’s new? Many tumor cells secrete proteases such as cathepsins, which are thought to enhance their ability to invade tissues and metastasize. V-ATPase inhibitors are known to inhibit metastases in mice. In this study, the authors tie these two lines of study together: They found that the V-ATPase inhibitor archazolid impairs the secretion of mature proteases such as cathepsin B and D, causing the proenzyme forms to be released instead. This represents a novel mechanism that may further explain the therapeutic effects of a promising new class of chemotherapeutic agents.
Material and Methods Compounds
Bafilomycin A1, concanamycin A, tunicamycin, brefeldin, NH4Cl, doxorubicin and chloroquine were purchased from Sigma Aldrich, archazolid A was purified and isolated as described previously,6 archazolid B was chemically synthesized by Roethle et al.36 Both compounds were dissolved in dimethyl sulfoxide (DMSO) and show similar bioactivity.15 For all in vitro experiments, archazolid B was used. For all in vivo experiments, archazolid A was used. Cell culture
T-24 (DSMZ ACC 376, provided by Barbara Mayer, Department of Surgery, Clinic Grosshadern, University of Munich, C 2013 UICC Int. J. Cancer: 134, 2478–2488 (2014) V
Germany) cells were cultured in McCoy’s modified 5A media (Biochrom, Germany) supplemented with 10% FCS Biochrom, Germany. 4T1luc cells (Caliper life science) were cultured in RPMI 1640 media (Invitrogen) supplemented with 10% FCS. MCF-7 cells (Cell line service, Germany) were cultured in DMEM 4.5 g/L glucose (Invitrogen) supplemented with 20% FCS. Cells were grown at 37 C, 5% CO2 in a humidified atmosphere. Cell viability assay (CellTiterGlo)
To analyze the cell viability of T-24 cells the CellTiterGlo (Promega, Germany) assay was applied. This assay detects the ATP content of a given sample, thus indicating the relative amount of metabolizing, living cells in an ATP-dependent luciferase reaction. The assay was performed according to the manufacturer’s instructions. Briefly, 5 3 104 cells per well were seeded in 96-well plates. Twenty-four hours after seeding cells were treated in FCS free media. After compound incubation, half of the volume of each well was replaced by the reaction buffer. The luminescence was recorded using a Luminometer Lumat LB9507 instrument (Berthold, Germany). Results were calculated as percent of control. Collection of conditioned media
1 3 106 MCF-7 or T24 cells were seeded in 10 cm dishes (Nunc). Twenty-four hours after seeding cells were washed three times (for proteomic analysis) or one time (western blot) with FCS-free media. After treatment of the cells in 10 mL of FCS-free media, supernatants were harvested, followed by centrifugation of 5 min at 1,000 rpm to remove cells and cell debris. Conditioned media samples were concentrated using Amicon Ultra-4 Centrifugal Filter Units (Merck, Millipore); cutoff 3 kDa for proteomics and 10 kDa for western blot. SDS-PAGE
SDS-PAGE was performed according to Laemmli et al.37 Conditioned media (30 mL) or 15–50 mg of total protein lysate were supplemented with Laemmli buffer and boiled for 5 min at 95 C for protein denaturation. Samples were loaded on 10 or 12.5% SDS-PA-gels. Protein separation was performed at 0.03 mA per gel for approximately 2 hr. In-gel-digestion
After electrophoresis, each lane was cut into 10 slices. Each piece was transferred into a reaction tube and covered with 45 mM DTT (Sigma Aldrich) in 50 mM NH4HCO3 (Sigma
Cancer Therapy
cathepsin B directly contribute to the matrix degradation.19–21 In contrast, others show that intracellular or membrane bound cathepsin B is crucial for extracellular matrix degradation by other proteases like urokinase (uPA) or matrix metalloproteinases.22,23 Nevertheless, cathepsin B expression is increased in different types of cancer like prostate cancer, oesophageal adenocarcinoma, breast cancer or colorectal cancer.24–30 Cathepsin B (CTSB) is synthesized at the rough endoplasmic reticulum as preprocathepsin B. Signal peptides target preproCTSB to the ER lumen, where it becomes cleaved to proCTSB. Subsequently, procathepsin B is transported through the ER to the Golgi apparatus. In the trans-Golgi network (TGN), it is bound after glycosylation and phosphorylation to membrane-associated mannose-6-phosphate receptor (M6PR) and proCTSB bound to M6PR containing vesicles are formed. These vesicles are directed to prelysosomal compartments (PLCs). During the lysosome maturation process, the pH of these PLC is decreasing which leads to the release of proCTSB from the M6PR and to the recycling of the receptor to the TGN. PLC are fusing with lysosomes and proCTSB is subsequently cleaved to active CTSB. Two forms of active CTSB are known: (i) the one chain form (31 kDA) and (ii) the two chain form (25/26 1 5 kDA).31–33 As extracellular signaling and cell–cell communications play an important role during metastatic processes34 and VATPase inhibitors modify endo- and exocytotic events by altering membrane trafficking15,35 the following hypothesis arouse: archazolid alters the secretion profile of invasive tumor cells contributing to its antimetastatic potential. We analyzed the secretome of archazolid-treated migratory carcinoma cells, found an impaired cathepsin B maturation process and evaluated its functional impact in vitro as well as in vivo.
2480
V-ATPase inhibition leads to impaired CTSB activation
Aldrich). After an incubation of 30 min at 55 C gel-pieces were covered with 100 mM iodoacteamide (Sigma Aldrich) in 50 mM NH4HCO3 for 15 min at RT in the dark. This step was repeated. Subsequently, gel-pieces were washed two time using 50 mM NH4HCO3. For tryptic digestion, pieces were chopped using a pipet tip and covered with 50 mM NH4HCO3 containing 70 ng trypsin (Promega, Germany) to 50 mg of protein. Incubation was carried out at 37 C overnight. After the digestion, supernatants were collected and pieces covered with 70% acetonitrile for at least 10 min. Supernatants were combined afterwards. After freeze-drying, peptide samples were stored at 280 C until LC-MS/MS measurement.
Cancer Therapy
LC-MS/MS analysis
Nano-LC separation was done with a nanoliquid chromatography system (Ettan MDLC, GE Healthcare). Peptide samples were loaded on a trap column (10 mL/min, loading buffer: 0.1% formic acid; trap column: C18 PepMap 100, 5-mm bead size, 300 mm i.d., 5 mm length, LC Packings) and separated with an analytical reversed phase column (Reprosil-Pur C18 AQ, 3 mm; 150 mm 3 75 mm, Dr Maisch, Germany) using a 30-min gradient from 0% B to 60% B (solvent A: 0.1% formic acid; solvent B: 84% CH3CN/0.1% formic acid), at a flow rate of 280 nL/min. For electrospray ionization, a distal coated SilicaTips (FS-360-20-10-D-20, New Objective) and a needle voltage of 1.7 kV was used. Tandem mass spectrometry was performed with an Orbitrap XL mass spectrometer (Thermo Scientific). MS and MS/MS spectra were acquired using cycles of one MS scan (mass range m/z 300–2000) and five subsequent data-dependent CID MS/MS scans (“dynamic exclusionTM activated”; collision energy: 35%). RAW data were processed using MASCOT Daemon and MASCOT Server (V2.3, Matrix Science, Boston) with the human subset of the SwissProt Database (Release 2012_11) and the following parameters: (i) “Fixed modifications”: Carbamidomethyl (C); (ii) Variable modifications: Oxidation (M); (iii) Decoy database: checked; (iv) Peptide charge: 11, 21 and 31; (v) Peptide tol. 6: 2 Da; (vi) MS/MS tol. 6: 0.8 Da. MASCOT data files were further evaluated using Scaffold 2.04 (Proteome Software), filtered identifications with at least two individual peptides (>95%) and a total false discovery rate
