308802) for tests were purchased from BioLedgend (Karrinyup, WA, Australia). IL-12p40 ELISA package from eBioscience (NORTH PARK, CA, USA). Statistical evaluation was performed from the two-tailed t-test.(TIF) pone.0213813.s004.tif (466K) GUID:?1D6C965B-B27C-4EC8-B766-7160C6D61A05 S1 Desk: Protein identification results of three biological replicates of U937 cells treated by LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody. For every replicate, you can find proteins assisting and determined peptides, iTRAQ quantified protein in support of peptides with ordinary local confidence higher than 80%.(XLSX) pone.0213813.s005.xlsx (1.3M) GUID:?42BAC5C8-C2A9-4A4F-8EC7-4DBDF33C78D6 S2 Desk: Proteins quantitation outcomes of three biological replicates of U937 cells treated by LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody, with regards to the control. For every replicate, there are iTRAQ labelled protein and regulated protein considerably.(XLSX) pone.0213813.s006.xlsx (655K) GUID:?5B7240A8-130C-4AD3-9689-EBE2463779AB S3 Desk: The gene oncology and PPIs of significantly controlled protein. (XLSX) pone.0213813.s007.xlsx (521K) GUID:?2C47091C-04F0-495F-84CD-51A7C7C9ABBF S4 Desk: The over-represented natural procedures and KEGG enriched related to upregulated protein of every treatment. (XLSX) pone.0213813.s008.xlsx (64K) GUID:?3A1B6402-3639-4F36-959B-0465E3ADDDB5 S1 Document: Other significant modulated canonical pathways identified from differentially expressed proteins identified from U937 cells using the treatments of LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody. (PDF) pone.0213813.s009.pdf (4.7M) GUID:?4B235916-1661-4EBD-BE2C-9CFD3A634AA5 Data Availability StatementAll relevant data are inside the manuscript and its own Supporting Info files. Abstract Blocking cytokine interleukin 10 (IL-10) during immunisation enhances vaccine induced T cell reactions and boosts control of tumour cell development immunisation was bought from BioXcell (USA) and kept at -80C till further make use of. Anti-IL-10 (Kitty. 506802), anti-IL-10R antibodies (Kitty. 308802) for Fenretinide tests had been purchased from BioLedgend (Karrinyup, WA, Australia). PE conjugated anti-IL-10R antibody (Kitty. 308803) was purchased from BioLegend. Cell iTRAQ and lysis labelling of peptides Unstimulated U937 cells were used while guide and cultured over night. U937 cells had been either activated with 410?3 M of LPS overnight (positive control) or treated with LPS plus 10 g/mL concentration of anti-IL-10 or anti-IL-10R antibodies, respectively. Following the treatment, cell pellets had been cleaned with 1 mL of cool PBS and counted, after that 1106 cells had been lysed with 300 l of lysis buffer (8M urea, 0.8M NH4HCO3, pH 8.0) supplemented with 10 l of protease inhibitor cocktail (80-6501-23, GE Healthcare, Small Chalfont, UK) to avoid protein degradation. The examples had been sonicated for 30 min on snow after that, and centrifuged at 12 after that,000 g at 4C for 15 min. The supernatants had been collected, and proteins focus in the cell lysates was assessed using the Pierce BCA proteins assay kit on the NanoDrop 2000 (Thermo Fisher Scientific, Bremen, Germany). After that, 500 g of protein had been decreased with 5 l of 100 mM DTT for 1 h at 37C, and consequently alkylated with 20 l of Fenretinide 100 mM IAA for 1 h at space temperature (RT) at night, accompanied by the incubation with the help of 20 L of 100 mM DTT at RT for 45 min. The urea focus was decreased to below 2M by diluting the response blend with MilliQ drinking water, then your proteins digested with sequencing quality customized trypsin (Promega, Madison, WI) at 1:50 enzyme\to\substrate percentage. After 4 h of digestive function at 37C, examples had been diluted 1:4 with 50 mM NH4HCO3, 1 mM CaCl2 and another aliquot from the same quantity of trypsin was put into the samples and additional incubated at space temperature over night (~16 h). The digested examples had been after that acidified with 10% FA to pH 3. Tryptic peptides had been desalted on Sep-Pak C18 columns (Waters, Milford, MA) and dried out using Speed-Vac concentrator (Thermo Fischer, Massachussets, USA). Peptides (100 g) from three examples had been dissolved in 30 L of 0.5 M triethylammonium bicarbonate, pH 8.5 solution, and blended with 1 units of iTRAQ reagent that was dissolved freshly in 70 L of ethanol. Route 114 was useful for labelling the research (unstimulated), 115 for labelling LPS activated cells, 116 and 117 for labelling LPS plus anti-IL-10 or anti-IL-10 receptor antibodies, respectively. After 1 h incubation at RT, 300 L of water was incubated and added for 30 min at RT to avoid the reaction. Peptides labelled by different iTRAQ reagents had been combined and focused to ~200 L after that, and had been desalted on C18 SPE columns, kept and dried out at -20 oC. Biological triplicates had been ready using the same process. NanoLC tandem TripleTof MS/MS analyses The iTRAQ labelled peptides had been resuspended in 25 L 0.1% FA.(XLSX) pone.0213813.s007.xlsx (521K) GUID:?2C47091C-04F0-495F-84CD-51A7C7C9ABBF S4 Desk: The over-represented biological procedures and KEGG enriched corresponding to upregulated protein of every treatment. gathered and assessed by IL-6 or IL-12p40 ELISA package from eBioscience (NORTH PARK, CA, USA). Statistical evaluation was performed with the two-tailed t-test.(TIF) pone.0213813.s004.tif (466K) GUID:?1D6C965B-B27C-4EC8-B766-7160C6D61A05 S1 Desk: Protein identification results of three biological replicates of U937 cells treated by LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody. For every replicate, a couple of protein discovered and helping peptides, iTRAQ quantified protein in support of peptides with standard local confidence higher than 80%.(XLSX) pone.0213813.s005.xlsx (1.3M) GUID:?42BAC5C8-C2A9-4A4F-8EC7-4DBDF33C78D6 S2 Desk: Proteins quantitation outcomes of three biological replicates of U937 cells treated by LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody, with regards to the control. For every replicate, there are iTRAQ labelled protein and significantly governed protein.(XLSX) pone.0213813.s006.xlsx (655K) GUID:?5B7240A8-130C-4AD3-9689-EBE2463779AB S3 Desk: The gene oncology and PPIs Fenretinide of significantly controlled protein. (XLSX) pone.0213813.s007.xlsx (521K) GUID:?2C47091C-04F0-495F-84CD-51A7C7C9ABBF S4 Desk: The over-represented natural procedures and KEGG enriched matching to upregulated protein of every treatment. (XLSX) pone.0213813.s008.xlsx (64K) GUID:?3A1B6402-3639-4F36-959B-0465E3ADDDB5 S1 Document: Other significant modulated canonical pathways identified from differentially expressed proteins identified from U937 cells using the treatments of LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody. (PDF) pone.0213813.s009.pdf (4.7M) GUID:?4B235916-1661-4EBD-BE2C-9CFD3A634AA5 Data Availability StatementAll relevant data are inside the manuscript and its own Supporting Details files. Abstract Blocking cytokine interleukin 10 (IL-10) during immunisation enhances vaccine induced T cell replies and increases control of tumour cell development immunisation was bought from BioXcell (USA) and kept at -80C till further make use of. Anti-IL-10 (Kitty. 506802), anti-IL-10R antibodies (Kitty. 308802) for tests had been purchased from BioLedgend (Karrinyup, WA, Australia). PE conjugated anti-IL-10R antibody (Kitty. 308803) was purchased from BioLegend. Cell lysis and iTRAQ labelling of peptides Unstimulated U937 cells had been used as guide and cultured right away. U937 cells had been either activated with 410?3 M of LPS overnight (positive control) or treated with LPS plus 10 g/mL concentration of anti-IL-10 or anti-IL-10R antibodies, respectively. Following the treatment, cell pellets had been cleaned with 1 mL of frosty PBS and counted, after that 1106 cells had been lysed with 300 l of lysis buffer (8M urea, 0.8M NH4HCO3, pH 8.0) supplemented with 10 l of protease inhibitor cocktail (80-6501-23, GE Healthcare, Small Chalfont, UK) to avoid proteins degradation. The examples had been after that sonicated for 30 min on glaciers, and centrifuged at 12,000 g at 4C for 15 min. The supernatants had been collected, and proteins focus in the cell lysates was assessed using the Pierce BCA proteins assay kit on the NanoDrop 2000 (Thermo Fisher Scientific, Bremen, Germany). After that, 500 g of protein had been decreased with 5 l of 100 mM DTT for 1 h at 37C, and eventually alkylated with 20 l of 100 mM IAA for 1 h at area temperature (RT) at night, accompanied by the incubation by adding 20 L of 100 mM DTT at RT for 45 min. The urea focus was decreased to below 2M by diluting the response mix with MilliQ drinking water, then your proteins digested with sequencing quality improved trypsin (Promega, Madison, WI) at 1:50 enzyme\to\substrate proportion. After 4 h of digestive function at 37C, examples had been diluted 1:4 with 50 mM NH4HCO3, 1 mM CaCl2 and another aliquot from the same quantity of trypsin was put into the samples and additional incubated at area temperature right away (~16 h). The digested examples had been after that acidified with 10% FA to pH 3. Tryptic peptides had been desalted on Sep-Pak C18 columns (Waters, Milford, MA) and dried out using Speed-Vac concentrator (Thermo Fischer, Massachussets, USA). Peptides (100 g) from three examples had been dissolved in 30 L of 0.5.PE conjugated anti-IL-10R antibody (Kitty. or IL-12p40 ELISA package from eBioscience (NORTH PARK, CA, USA). Statistical evaluation was performed with the two-tailed t-test.(TIF) pone.0213813.s004.tif (466K) GUID:?1D6C965B-B27C-4EC8-B766-7160C6D61A05 S1 Desk: Protein identification results of three biological replicates of U937 cells treated by LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody. For every replicate, a couple of protein discovered and helping peptides, iTRAQ quantified protein in support of peptides with standard local confidence higher than 80%.(XLSX) pone.0213813.s005.xlsx (1.3M) GUID:?42BAC5C8-C2A9-4A4F-8EC7-4DBDF33C78D6 S2 Desk: Proteins quantitation outcomes of three biological replicates of U937 cells treated by LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody, with regards to the control. For every replicate, there are iTRAQ labelled protein and significantly governed protein.(XLSX) pone.0213813.s006.xlsx (655K) GUID:?5B7240A8-130C-4AD3-9689-EBE2463779AB S3 Desk: The gene oncology and PPIs of significantly controlled protein. (XLSX) pone.0213813.s007.xlsx (521K) GUID:?2C47091C-04F0-495F-84CD-51A7C7C9ABBF S4 Desk: The over-represented natural procedures and KEGG enriched matching to upregulated protein of every treatment. (XLSX) pone.0213813.s008.xlsx (64K) GUID:?3A1B6402-3639-4F36-959B-0465E3ADDDB5 S1 Document: Other significant modulated canonical pathways identified from differentially expressed proteins identified from U937 cells using the treatments of LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody. (PDF) pone.0213813.s009.pdf (4.7M) GUID:?4B235916-1661-4EBD-BE2C-9CFD3A634AA5 Data Availability StatementAll relevant data are inside the manuscript and its own Supporting Details files. Abstract Blocking cytokine interleukin 10 (IL-10) during immunisation enhances vaccine induced T cell replies and increases control of tumour cell development immunisation was bought from BioXcell (USA) and kept at -80C till further make use of. Anti-IL-10 (Kitty. 506802), anti-IL-10R antibodies (Kitty. 308802) for tests had been purchased from BioLedgend (Karrinyup, WA, Australia). PE conjugated anti-IL-10R antibody (Kitty. 308803) was purchased from BioLegend. Cell lysis and iTRAQ labelling of peptides Unstimulated U937 cells had been used as guide and cultured right away. U937 cells had been either activated with 410?3 M of LPS overnight (positive control) or treated with LPS plus 10 g/mL concentration of anti-IL-10 or anti-IL-10R antibodies, respectively. Following the treatment, cell pellets had been cleaned with 1 mL of frosty PBS and counted, after that 1106 cells had been lysed with 300 l of lysis buffer (8M urea, 0.8M NH4HCO3, pH 8.0) supplemented with 10 l of protease inhibitor cocktail (80-6501-23, GE Healthcare, Small Chalfont, UK) to avoid proteins degradation. The examples had been after that sonicated for 30 min on glaciers, and centrifuged at 12,000 g at 4C for 15 min. The supernatants had been collected, and proteins focus in the cell lysates was assessed using the Pierce BCA proteins assay kit on the NanoDrop 2000 (Thermo Fisher Scientific, Bremen, Germany). After that, 500 g of protein had been decreased with 5 l of 100 mM DTT for 1 h at 37C, and eventually alkylated with 20 l of 100 mM IAA for 1 h at area temperature (RT) at night, accompanied by the incubation by adding 20 L of 100 mM DTT at RT for 45 min. The urea focus was decreased to below 2M by diluting the response mix with MilliQ drinking water, then your proteins digested with sequencing grade altered trypsin (Promega, Madison, WI) at 1:50 enzyme\to\substrate percentage. After 4 h of digestion at 37C, samples were diluted 1:4 with 50 mM NH4HCO3, 1 mM CaCl2 and another aliquot of the same amount of trypsin was added to the samples and further incubated at space temperature immediately (~16 h). The digested samples were then acidified with 10% FA to pH 3. Tryptic peptides were desalted on Sep-Pak C18 columns (Waters, Milford, MA) and dried using Speed-Vac concentrator (Thermo Fischer, Massachussets, USA). Peptides (100 g) from three samples were dissolved in 30 L of 0.5 M triethylammonium bicarbonate, pH 8.5 solution, and mixed with 1 units of iTRAQ reagent that was dissolved freshly in 70 L of ethanol. Channel 114 was utilized for labelling the research (unstimulated), 115 for labelling LPS stimulated cells, 116 and 117.(PDF) pone.0213813.s009.pdf (4.7M) GUID:?4B235916-1661-4EBD-BE2C-9CFD3A634AA5 Data Availability StatementAll relevant data are within the manuscript and its Supporting Information documents. Abstract Blocking cytokine interleukin 10 (IL-10) at the time of immunisation enhances vaccine induced T cell responses and enhances control of tumour cell growth immunisation was purchased from BioXcell (USA) and stored at -80C till further use. Fig: The gene ontology analysis of significantly controlled proteins recognized in LPS+anti-IL-10R antibody treated U937 cells by iTRAQ-label LC-MS/MS. (TIF) pone.0213813.s003.tif (16M) GUID:?4B449F0A-1DA5-47CF-A483-595C91C23128 S4 Fig: 5105 of mouse PBMCs (A and B) or 5105 U937 cells (C) were either remaining untreated (UN) or stimulated with 100 ng of LPS, in the presence or absence of anti-IL-10 or anti-IL10R antibodies and cultured overnight. Supernatants were collected and measured by IL-6 or IL-12p40 ELISA kit from eBioscience (San Diego, CA, APRF USA). Statistical analysis was performed from the two-tailed t-test.(TIF) pone.0213813.s004.tif (466K) GUID:?1D6C965B-B27C-4EC8-B766-7160C6D61A05 S1 Table: Protein identification results of three biological replicates of U937 cells treated by LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody. For each replicate, you will find protein recognized and assisting peptides, iTRAQ quantified proteins and only peptides with common local confidence greater than 80%.(XLSX) pone.0213813.s005.xlsx (1.3M) GUID:?42BAC5C8-C2A9-4A4F-8EC7-4DBDF33C78D6 S2 Table: Protein quantitation results of three biological replicates of U937 cells treated by LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody, with respect to the control. For each replicate, there are all iTRAQ labelled proteins and significantly controlled proteins.(XLSX) pone.0213813.s006.xlsx (655K) GUID:?5B7240A8-130C-4AD3-9689-EBE2463779AB S3 Table: The gene oncology and PPIs of significantly regulated proteins. (XLSX) pone.0213813.s007.xlsx (521K) GUID:?2C47091C-04F0-495F-84CD-51A7C7C9ABBF S4 Table: The over-represented biological processes and KEGG enriched related to upregulated proteins of each treatment. (XLSX) pone.0213813.s008.xlsx (64K) GUID:?3A1B6402-3639-4F36-959B-0465E3ADDDB5 S1 File: Other significant modulated canonical pathways identified from differentially expressed proteins identified from U937 cells with the treatments of LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody. (PDF) pone.0213813.s009.pdf (4.7M) GUID:?4B235916-1661-4EBD-BE2C-9CFD3A634AA5 Data Availability StatementAll relevant data are within the manuscript and its Supporting Info files. Abstract Blocking cytokine interleukin 10 (IL-10) at the time of immunisation enhances vaccine induced T cell reactions and enhances control of tumour cell growth immunisation was purchased from BioXcell (USA) and stored at -80C till further use. Anti-IL-10 (Cat. 506802), anti-IL-10R antibodies (Cat. 308802) for experiments were purchased from BioLedgend (Karrinyup, WA, Australia). PE conjugated anti-IL-10R antibody (Cat. 308803) was purchased from BioLegend. Cell lysis and iTRAQ labelling of peptides Unstimulated U937 cells were used as research and cultured over night. U937 cells were either stimulated with 410?3 M of LPS overnight (positive control) or treated with LPS plus 10 g/mL concentration of anti-IL-10 or anti-IL-10R antibodies, respectively. After the treatment, cell pellets were washed with 1 mL of chilly PBS and counted, then 1106 cells were lysed with 300 l of lysis buffer (8M urea, 0.8M NH4HCO3, pH 8.0) Fenretinide supplemented with 10 l of protease inhibitor cocktail (80-6501-23, GE Healthcare, Little Chalfont, UK) to prevent protein degradation. The samples were then sonicated for 30 min on snow, and then centrifuged at 12,000 g at 4C for 15 min. The supernatants were collected, and protein concentration in the cell lysates was measured using the Pierce BCA protein assay Fenretinide kit on a NanoDrop 2000 (Thermo Fisher Scientific, Bremen, Germany). Then, 500 g of proteins were reduced with 5 l of 100 mM DTT for 1 h at 37C, and consequently alkylated with 20 l of 100 mM IAA for 1 h at space temperature (RT) in the dark, followed by the incubation with the help of 20 L of 100 mM DTT at RT for 45 min. The urea concentration was reduced to below 2M by diluting the reaction mixture with MilliQ water, then the proteins digested with sequencing grade modified trypsin (Promega, Madison, WI) at 1:50 enzyme\to\substrate ratio. After 4 h of digestion at 37C, samples were diluted 1:4 with 50 mM NH4HCO3, 1 mM CaCl2 and another aliquot of the same amount of trypsin was added to the samples and further incubated at room temperature overnight (~16 h). The digested samples were then acidified with 10% FA to pH 3. Tryptic peptides were desalted on Sep-Pak C18 columns (Waters, Milford, MA) and dried using Speed-Vac concentrator (Thermo Fischer, Massachussets, USA). Peptides (100 g) from three samples were dissolved in 30 L of 0.5 M triethylammonium bicarbonate, pH 8.5 solution, and mixed with 1 units of iTRAQ reagent that was dissolved freshly in 70 L of ethanol. Channel 114 was used for labelling the reference (unstimulated), 115 for labelling LPS stimulated cells, 116 and 117 for labelling LPS plus anti-IL-10 or anti-IL-10 receptor antibodies, respectively. After 1 h incubation at RT, 300 L of water was added and incubated for 30 min at RT to stop the reaction. Peptides labelled by different iTRAQ reagents were then mixed and concentrated to ~200 L, and were desalted on C18 SPE columns, dried and stored at -20 oC. Biological triplicates were prepared using the same protocol. NanoLC tandem TripleTof MS/MS analyses The iTRAQ labelled peptides were resuspended in 25 L 0.1% FA in MilliQ water and analysed by LC-MS/MS.The comparison among the triplicates showed that 585 proteins were mutually identified (Fig 1B). in the presence or absence of anti-IL-10 or anti-IL10R antibodies and cultured overnight. Supernatants were collected and measured by IL-6 or IL-12p40 ELISA kit from eBioscience (San Diego, CA, USA). Statistical analysis was performed by the two-tailed t-test.(TIF) pone.0213813.s004.tif (466K) GUID:?1D6C965B-B27C-4EC8-B766-7160C6D61A05 S1 Table: Protein identification results of three biological replicates of U937 cells treated by LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody. For each replicate, there are protein identified and supporting peptides, iTRAQ quantified proteins and only peptides with average local confidence greater than 80%.(XLSX) pone.0213813.s005.xlsx (1.3M) GUID:?42BAC5C8-C2A9-4A4F-8EC7-4DBDF33C78D6 S2 Table: Protein quantitation results of three biological replicates of U937 cells treated by LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody, with respect to the control. For each replicate, there are all iTRAQ labelled proteins and significantly regulated proteins.(XLSX) pone.0213813.s006.xlsx (655K) GUID:?5B7240A8-130C-4AD3-9689-EBE2463779AB S3 Table: The gene oncology and PPIs of significantly regulated proteins. (XLSX) pone.0213813.s007.xlsx (521K) GUID:?2C47091C-04F0-495F-84CD-51A7C7C9ABBF S4 Table: The over-represented biological processes and KEGG enriched corresponding to upregulated proteins of each treatment. (XLSX) pone.0213813.s008.xlsx (64K) GUID:?3A1B6402-3639-4F36-959B-0465E3ADDDB5 S1 File: Other significant modulated canonical pathways identified from differentially expressed proteins identified from U937 cells with the treatments of LPS, LPS+anti-IL-10 antibody and LPS+anti-IL-10R antibody. (PDF) pone.0213813.s009.pdf (4.7M) GUID:?4B235916-1661-4EBD-BE2C-9CFD3A634AA5 Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract Blocking cytokine interleukin 10 (IL-10) at the time of immunisation enhances vaccine induced T cell responses and improves control of tumour cell growth immunisation was purchased from BioXcell (USA) and stored at -80C till further use. Anti-IL-10 (Cat. 506802), anti-IL-10R antibodies (Cat. 308802) for experiments were purchased from BioLedgend (Karrinyup, WA, Australia). PE conjugated anti-IL-10R antibody (Cat. 308803) was purchased from BioLegend. Cell lysis and iTRAQ labelling of peptides Unstimulated U937 cells were used as reference and cultured overnight. U937 cells were either stimulated with 410?3 M of LPS overnight (positive control) or treated with LPS plus 10 g/mL concentration of anti-IL-10 or anti-IL-10R antibodies, respectively. After the treatment, cell pellets were washed with 1 mL of cold PBS and counted, then 1106 cells were lysed with 300 l of lysis buffer (8M urea, 0.8M NH4HCO3, pH 8.0) supplemented with 10 l of protease inhibitor cocktail (80-6501-23, GE Healthcare, Little Chalfont, UK) to prevent protein degradation. The samples were then sonicated for 30 min on ice, and then centrifuged at 12,000 g at 4C for 15 min. The supernatants were collected, and protein concentration in the cell lysates was measured using the Pierce BCA protein assay kit on a NanoDrop 2000 (Thermo Fisher Scientific, Bremen, Germany). Then, 500 g of proteins were reduced with 5 l of 100 mM DTT for 1 h at 37C, and subsequently alkylated with 20 l of 100 mM IAA for 1 h at room temperature (RT) in the dark, followed by the incubation with the addition of 20 L of 100 mM DTT at RT for 45 min. The urea concentration was reduced to below 2M by diluting the reaction mixture with MilliQ water, then the proteins digested with sequencing grade modified trypsin (Promega, Madison, WI) at 1:50 enzyme\to\substrate ratio. After 4 h of digestion at 37C, samples were diluted 1:4 with 50 mM NH4HCO3, 1 mM CaCl2 and another aliquot of the same amount of trypsin was added to the samples and further incubated at room temperature overnight (~16 h). The digested samples were then acidified with 10% FA to pH 3. Tryptic peptides were desalted on Sep-Pak C18 columns (Waters, Milford, MA) and dried using Speed-Vac concentrator (Thermo Fischer, Massachussets, USA). Peptides (100 g) from three samples were dissolved in 30 L of 0.5 M triethylammonium bicarbonate, pH.