Project description:Multiple Affinity Removal Spin Cartridge Human 14 kit (Agilent) was used to remove 14 major proteins from plasma. Samples were denatured with an equivalent volume of trifluoroethanol and reduced with dithiothreitol. Free cysteine residues were alkylated with iodoacetamide. Samples were incubated with trypsin and desalted with C18 ZipTip. Desalted samples were rehydrated in 0.1% formic acid (FA) and were analyzed by LC-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to an TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using liquid chromatography with a trap and elution conﬁguration using a nano trap column (350 μm × 0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse phase column (75 μm × 15 cm, 3 μm, 120 Å, AB Sciex) at 300 nl/min with a 90 min linear gradient of 8-30% acetonitrile in 0.1% FA, and then, with a 10 min linear gradient of 30% to 40% acetonitrile in 0.1% FA. The mass spectrometer was operated in information-dependent acquisition (IDA) mode, scanning full spectra (400–1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100–1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and + 5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. Rolling collision energy was used with a collision energy spread of 15. The mass spectrometer was operated using the Analyst TF 1.7.1 software program (AB Sciex). For data dependent acquisition (DDA, SWATH acquisition), the parameters were set as follows: 100 ms TOF MS scan, followed by 200 variable SWATH windows each at 50 ms accumulation time for m/z 400–1250. MS/MS SWATH scans were set at 5 Da window overlapping by 1 Da for m/z 400–1250 and varied on each side of the mass range. The total cycle time was 9.6 s. A rolling collision energy (CE) parameters script was used to automatically control the CE. Acquired spectra were searched against the UniProt reviewed database using the Paragon algorithm embedded in the ProteinPilot 5.0.1 software program (AB Sciex), with the following search parameters: (i) sample type: identiﬁcation, (ii) Cys alkylation: iodoacetamide, (iii) digestion: trypsin, (iv) instrument: TripleTOF 6600, (v) species: Homo sapiens, (vi) ID focus: biological modiﬁcations, (vii) detected protein threshold: > 0.05 (10% conﬁdence). The detected protein threshold was set to the minimum level to enhance the number of wrong answers to enable the curve ﬁtting by an independent FDR analysis. This was carried out by the target-decoy approach provided with the ProteinPilot software program, which was used to assess the quality of the identiﬁcations. Positive identiﬁcations were considered when identiﬁed proteins and peptides reached a 1% local FDR. The resulting group file was loaded into Peakview (v2.2.0, AB Sciex) and peaks from SWATH runs were extracted with a peptide confidence threshold of 99% and a false discovery rate <1%. The SWATH ﬁles were then exported to the MarkerView software program (version 1.3.0.1; AB Sciex) and the peak areas of individual peptides were normalized to the sum of the peak areas of all detected peptides.

Project description:The precipitated proteins from whole cells were resuspended in 50 μl of 500 mM ammonium bicarbonate containing 8 M urea, and reduced with 5 μl of 200 mM DTT. The samples were incubated at 90°C for 30 min and cooled to room temperature. Free cysteine residues were alkylated with 10 μl of 200 mM iodoacetamide for 60 min at room temperature in the dark, and the remaining iodoacetamide was quenched by adding 5 μl of 200 mM DTT. The samples were then mixed with 320 μl of 100 mM ammonium bicarbonate. The samples were incubated with 5 μg trypsin (AB Sciex) at 37°C for 18 h. The samples were desalted with C18 ZipTip (Millipore, Bedford, MA, USA) and eluted with H2O/acetonitrile (5/5; v/v). The ZipTip eluates were dried in a vacuum centrifuge. Desalted samples were rehydrated in 0.1% formic acid and analyzed with liquid chromatography (LC)-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to a TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using LC on a trap and elution conﬁguration using a nano trap column (350 μm × 0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse-phase column (75 μm × 15 cm, 3 μm, 120 Å, AB Sciex) at 300 nl/min with a 90-min linear gradient of 8% to 30% acetonitrile in 0.1% formic acid, and then, with a 10-min linear gradient of 30% to 40% acetonitrile in 0.1% formic acid. The MS was operated in information-dependent acquisition mode, scanning full spectra (400–1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100–1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and +5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. Rolling collision energy (CE) was used with a CE spread of 15. The MS was operated by Analyst TF 1.7.1 software (AB Sciex). For data-independent acquisition (SWATH acquisition), the parameters were set as follows: 100 ms TOF MS scan, followed by 200 variable SWATH windows each at a 50-ms accumulation time for m/z 400–1250. MS/MS SWATH scans were set at 5-Da windows that overlapped by 1 Da for m/z 400–1250 and varied on each side of the mass range. The total cycle time was 9.6 s. Rolling CE parameter script was used for automatically controlling the CE. Data Analysis—Acquired spectra were searched against the UniProt reviewed database using the Paragon algorithm embedded in ProteinPilot 5.0.1 software (AB Sciex), with the following search parameters: (i) sample type: identiﬁcation, (ii) Cys alkylation: iodoacetamide, (iii) digestion: trypsin, (iv) instrument: TripleTOF 6600, (v) species: Mus musculus, (vi) ID focus: biological modiﬁcations, (vii) detected protein threshold: >0.05 (10% conﬁdence). The detected protein threshold was set to the minimum level to enhance the number of wrong answers to enable the curve ﬁtting by an independent false discovery rate (FDR) analysis45. This was carried out by the target-decoy approach provided with ProteinPilot software, which was used to assess the quality of the identiﬁcations. Identiﬁcations were considered positive when identiﬁed proteins and peptides reached a 1% local FDR46. The resulting .group file was loaded into Peakview (v2.2.0, AB Sciex), and peaks from SWATH runs were extracted with a peptide confidence threshold of 99% and a FDR <1%. The SWATH ﬁles were then exported to MarkerView software (version 1.3.0.1; AB Sciex), and peak areas of individual peptides were normalized to the sum of peak areas of all detected peptides. Statistics of PCA and OPLS-DA were performed using Simca software (Infocom Corp., Tokyo, Japan) for multivariate statistical analysis. Pareto scaling was applied to the peak area values acquired by SWATH prior to the analyses, which represents a compromise between the extremes of no scaling and unit variance scaling and involves dividing each spectral variable by the square root of its standard deviation after first centering.

Project description:Concentration of extracted proteins were determined by BCA method using albumin as a standard. Sample volume was adjusted so that the each sample contain 20 μg protein, and proteins were precipitated with chilled acetone. The pellet was resuspended with 40 μl of 50 mM ammonium bicarbonate containing 50% trifluoroethanol and reduced with 1 μl of 200 mM dithiothreitol (DTT). The samples were incubated at 90°C for 30 min and cooled to room temperature. Free cysteine residues were alkylated with 4 μl of 200 mM iodoacetamide for 60 min at room temperature in the dark and the remaining iodoacetamide was quenched by adding 1 μl of 200 mM DTT. The samples were then diluted with 300 μl of 50 mM ammonium bicarbonate and incubated with 1 μg trypsin (TPCK treated, AB Sciex, Framingham, MA, USA) at 37 °C for 18 h. The samples were desalted with C18 ZipTip (Millipore, Bedford, MA, USA) and eluted with H2O/acetonitrile (5/5; v/v). The ZipTip eluates were dried in a vacuum centrifuge. Desalted samples were rehydrated in 0.1% formic acid (FA) and were analyzed by LC-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to an TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using liquid chromatography with a trap and elution conﬁguration using a nano trap column (350 μm × 0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse phase column (75 μm × 15 cm, 3 μm, 120 Å, AB Sciex) at 300 nl/min with a 90 min linear gradient of 8-30% acetonitrile in 0.1% FA, and then, with a 10 min linear gradient of 30% to 40% acetonitrile in 0.1% FA. The mass spectrometer was operated in information-dependent acquisition (IDA) mode, scanning full spectra (400–1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100–1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and + 5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. Rolling collision energy was used with a collision energy spread of 15. The mass spectrometer was operated using the Analyst TF 1.7.1 software program (AB Sciex). For data dependent acquisition (DDA, SWATH acquisition), the parameters were set as follows: 100 ms TOF MS scan, followed by 200 variable SWATH windows each at 50 ms accumulation time for m/z 400–1250. MS/MS SWATH scans were set at 5 Da window overlapping by 1 Da for m/z 400–1250 and varied on each side of the mass range. The total cycle time was 9.6 s. A rolling collision energy (CE) parameters script was used to automatically control the CE. Acquired spectra were searched against the UniProt reviewed database using the Paragon algorithm embedded in the ProteinPilot 5.0.1 software program (AB Sciex), with the following search parameters: (i) sample type: identiﬁcation, (ii) Cys alkylation: iodoacetamide, (iii) digestion: trypsin, (iv) instrument: TripleTOF 6600, (v) species: Homo sapiens, (vi) ID focus: biological modiﬁcations, (vii) detected protein threshold: > 0.05 (10% conﬁdence). The detected protein threshold was set to the minimum level to enhance the number of wrong answers to enable the curve ﬁtting by an independent FDR analysis(1). This was carried out by the target-decoy approach provided with the ProteinPilot software program, which was used to assess the quality of the identiﬁcations. Positive identiﬁcations were considered when identiﬁed proteins and peptides reached a 1% local FDR(2). The resulting group file was loaded into Peakview (v2.2.0, AB Sciex) and peaks from SWATH runs were extracted with a peptide confidence threshold of 99% and a false discovery rate <1%. The SWATH ﬁles were then exported to the MarkerView software program (version 1.3.0.1; AB Sciex) and the peak areas of individual peptides were normalized to the sum of the peak areas of all detected peptides.

Project description:Mitochondrial fraction was obtained using the mitochondrial isolation kit (KC010100, BioChain Institute). Each tissue was finely chopped and homogenized 40 strokes in two volumes of mitochondrial isolation buffer, and then centrifuged at 600×g for 10 min. The supernatant was collected and centrifuged at 12,000×g for 15 min. The supernatant was used as cytosolic fraction containing lysosomes and microsomes. The mitochondrial fraction was obtained from the pellet and resuspended in mitochondrial isolation buffer. The suspended mitochondria were sonicated (TOMY, Ultrasonic disruptor UD-100, output level 30, 30 sec×2) and centrifuged at 105,000×g for 30 min. The supernatant was used as the mitochondrial soluble fractions, containing mitochondrial intermembrane space (IMS) and matrix (MAT). The pellet was resuspended in RIPA buffer and centrifuged at 20,000×g for 30 min. The supernatant was used as the mitochondrial membrane fractions, containing mitochondrial outer membrane (OM) and inner membrane (IM). All the procedures were performed at 4°C. Mitochondrial and cytosolic fractions of WT and ES1-KO mouse brain were isolated as described 2.1 in 1% triton x-100 in 20 mM Tris-HCl. These samples were concentrated by SDS-PAGE and cut out from gels. The sectioned gels were finely chopped and washed with water. Then, the gel pieces were destained 50% methanol and vortexed in 25 mM ammonium bicarbonate containing 50% acetonitrile for 5 min. The supernatant was removed and the gel pieces were soaked 100% acetonitrile for 1 min. After removing the solution, the gel pieces were dried in vacuo, and rehydrated with a reducing solution (50 mM ammonium bicarbonate and 25 mM dithiothreitol), and incubated for 40 min at 56°C. The reducing buffer was removed and gel pieces were soaked in an alkylating solution (50 mM ammonium bicarbonate containing 55 mM iodoacetamide) for 30 min at room temperature in the dark. After removing the alkylation solution, the gel pieces were washed twice with water and vortexed in 25 mM ammonium bicarbonate containing 50% acetonitrile for 5 min, and then soaked 100% acetonitrile for 1 min. After removing the solution, the gel pieces were dried in vacuo and rehydrated with 50 mM ammonium bicarbonate containing 4 ng/μL sequencing grade trypsin and 0.01% Protease Max Surfactant. After incubating 10 min on ice, an equivalent volume of 50 mM ammonium bicarbonate containing 0.01% Protease Max Surfactant was added and incubated for 3 h at 37°C to digest the proteins in the gels with a protease. The equivalent volume of 1% trifluoroacetic acid was added and vortexed for 10 min for extraction of the tryptic fragments. After centrifuged at 12,000×g for 15 min, the peptide solutions were obtained from the supernatant. Ten microliters of each peptide solution were diluted with 190 μL of 100 mM ammonium bicarbonate and filtered on with a 0.22 μm using the amicon ultrafiltration unit (Amicon Ultra, 3 kDa, Millipore, MA, USA). Concentrated samples were denatured with an equivalent volume of trifluoroethanol (25 μL) and reduced with 1 μL of 200 mM dithiothreitol (DTT). The samples were incubated at 90°C for 30 min and cooled to room temperature. Free cysteine residues were alkylated with 4 μL of 200 mM iodoacetamide for 60 min at room temperature in the dark and the remaining iodoacetamide was quenched by adding 1 μL of 200 mM DTT. The samples were then mixed with 300 μL of 100 mM ammonium bicarbonate. Fifteen microliters of the sample were diluted with 85 μL of 100 mM ammonium bicarbonate and incubated with 1 μg trypsin (TPCK treated, AB Sciex, Framingham, MA, USA) at 37 °C for 18 h. The samples were desalted with C18 ZipTip (Millipore, Bedford, MA, USA) and eluted with H2O/acetonitrile (5/5; v/v). The ZipTip eluates were dried in a vacuum centrifuge. Desalted samples were rehydrated in 0.1% formic acid (FA) and were analyzed by LC-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to a TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using liquid chromatography with a trap and elution conﬁguration using a nano trap column (350 μm×0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse-phase column (75 μm×15 cm, 3 μm, 120 Å, AB Sciex) at 300 nL/min with a 90 min linear gradient of 8-30% acetonitrile in 0.1% FA, and then, with a 10 min linear gradient of 30% to 40% acetonitrile in 0.1% FA. The mass spectrometer was operated in information-dependent acquisition (IDA) mode, scanning full spectra (400–1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100–1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and + 5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. The rolling collision energy was used with a collision energy spread of 15. The mass spectrometer was operated using the Analyst TF 1.7.1 software program (AB Sciex). For data-dependent acquisition (DDA, SWATH acquisition), the parameters were set as follows: 100 ms TOF MS scan, followed by 200 variable SWATH windows each at 50 ms accumulation time for m/z 400–1250. MS/MS SWATH scans were set at 5 Da window overlapping by 1 Da for m/z 400–1250 and varied on each side of the mass range. The total cycle time was 9.6 s. Rolling collision energy (CE) parameter script was used to automatically control the CE. Acquired spectra were searched against the UniProt reviewed database using the Paragon algorithm embedded in the ProteinPilot 5.0.1 software program (AB Sciex), with the following search parameters: (i) sample type: identiﬁcation, (ii) Cys alkylation: iodoacetamide, (iii) digestion: trypsin, (iv) instrument: TripleTOF 6600, (v) species: Mus musculus, (vi) ID focus: biological modiﬁcations, (vii) detected protein threshold: > 0.05 (10% conﬁdence). The detected protein threshold was set to the minimum level to enhance the number of wrong answers to enable the curve ﬁtting by an independent FDR analysis. This was carried out by the target-decoy approach provided with the ProteinPilot software program, which was used to assess the quality of the identiﬁcations. Positive identiﬁcations were considered when identiﬁed proteins and peptides reached a 1% local FDR. The resulting group file was loaded into Peakview (v2.2.0, AB Sciex) and peaks from SWATH runs were extracted with a peptide confidence threshold of 99% and a false discovery rate <1%. The SWATH ﬁles were then exported to the MarkerView software program (version 1.3.0.1; AB Sciex) and the peak areas of individual peptides were normalized to the sum of the peak areas of all detected peptides.

			Action	DRS
	OVC_WT-3_IDA.wiff	Wiff
	OVC_WT-3_IDA.wiff.scan	Wiff
	OVC_hCBR1-tGFP_No.1-1_IDA.wiff	Wiff
	OVC_hCBR1-tGFP_No.1-1_IDA.wiff.scan	Wiff
	OVC_hCBR1-tGFP_No.1-2_IDA.wiff	Wiff

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Proteome of CBR1 transgenic SK-OV-3 and OVCAR-2 cells

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