Pancreatic cancer proteome from paraffin-embedded tissue
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: Kenichi Hakamada
PROVIDER: PXD025975 | JPOST Repository | Wed Oct 27 00:00:00 BST 2021
REPOSITORIES: jPOST
Publications
Eukaryotic initiation factor 2 signaling behind neural invasion linked with lymphatic and vascular invasion in pancreatic cancer.
Wakiya Taiichi T Ishido Keinosuke K Kimura Norihisa N Nagase Hayato H Yoshizawa Tadashi T Morohashi Satoko S Fujita Hiroaki H Kanda Taishu T Tatara Yota Y Saruwatari Junji J Kijima Hiroshi H Hakamada Kenichi K
Scientific reports 20211027 1
Perineural invasion (PNI) is a typical poor prognostic factor in pancreatic ductal adenocarcinoma (PDAC). The mechanisms linking PNI to poor prognosis remain unclear. This study aimed to clarify what changes occurred alongside PNI in PDAC. A 128-patient cohort undergoing surgery for early-stage PDAC was evaluated. Subdivided into two groups, according to pathological state, a pancreatic nerve invasion (ne) score of less than three (from none to moderate invasion) was designated as the low-grade ...[more]
Similar Datasets
Project description:Flag-YBX1 overexpressed T24 cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15 000 g for 20 min. Before incubating the lysate with Flag beads, 100ul were taken as input. The anti-Flag M2 magnetic beads (Sigma, 10 μl per mg lysate) were washed with NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 h with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1 000 000 dilution) for 8 min at 37 °C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50 °C) Proteinase K and RNAs were extracted with an equal volume of Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to rRNA removal and Bisseq. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length.The m5C sites were called using meRanCall from meRanTK (FDR < 0.01).
2019-07-02 | GSE133625 | GEO
Project description:10 µm formalin-fixed paraffin-embedded tissue sections were deparaffinized with xylene; homogenization was performed in lysis-buffer (20 mM Tris-HCl, pH 8.8, 200 mM glycine, 200 mM DTT, 4% SDS)in an ultrasonic bath; samples were incubated under continuous agitation first at 99 °C for 30 min, then at 80 °C for 60 min, afterwards centrifuged at 12,000xg for 10 min. 50 µg of protein lysate were further processed according the GASP (Gel-assisted sample preparation) protocol; peptide identification (IDA, information dependent acquisition) and SWATH measurements by LC-MS/MS on TripleTOF5600 (AB Sciex)
2020-05-18 | PXD012317 | Pride
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.
2022-05-20 | PXD026109 | JPOST Repository
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.
2022-05-12 | PXD025953 | JPOST Repository
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.
2022-05-12 | PXD025963 | JPOST Repository
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.
2022-05-12 | PXD025954 | JPOST Repository
Project description:Two sampling areas were focused in this study; Tande tande hamlet as high-level natural background radiation area (HBRA) and Topoyo sub-district as normal level background radiation area (NBRA), which was done under coordination with Local Health Office. The former located in 2° 44’ 38.3’’ south latitude/ 118° 48’ 47’’ east longitude belongs to Northern Botteng village in Simboro district of Mamuju city, and the population of the hamlet is 350 people in 2017 on an area of 16.22 km2 according to the information of BPS-Statistics of Mamuju Regency 2018. The latter located in 1° 59’ 38.3’’ south latitude/ 119° 28’ 16.7’’ east longitude belongs to Topoyo sub-district of Mamuju Tengah city about 120 km from Mamuju city, and the population of the sub-district is 31,888 people with 16,463 men (51.63%) and 15,425 women (48.37%) in 2017 on an area of 844.80 km2 according to the information of BPS-Statistics of Mamuju Regency 2019. 26 healthy adult inhabitants (13 males and 13 females) from Tande tande hamlet and 23 healthy adult inhabitants (14 males and 9 females) from Topoyo sub-district were included in this study. All volunteers were informed about the nature, aims, and intention of the study and signed a consent form and questionnaire to obtain information on age and occupation and another biodata before providing blood samples. Any individuals suffering from an illness or taking medication were excluded. Peripheral blood samples then were obtained from the antecubital vein using BD Vacutainer tubes in the morning (about from 6 a.m. to 9 a.m.) and placed at room temperature for at least 30 min to allow blood-clotting. Sera was collected by centrifugation at 1,200 ×g for 10 min at 4°C and stored at - 80°C until the analysis.
2 μl of sera was diluted with 198 μl of 50 mM ammonium bicarbonate. The serum proteins were precipitated by adding 1 ml of acetone. The precipitated proteins were collected by centrifugation at 15,000 ×g for 10 min. The precipitate was resuspended in 10 μl of 500 mM ammonium bicarbonate and denatured with an equivalent volume of trifluoroethanol and 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 (TPCK treated, 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 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 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.
On the basis of the result of the information-dependent acquisition, an assay for a high-resolution multiple reaction monitoring (MRM-HR) experiment targeted to the serum albumin oxidative modification was developed using Skyline software (MacCoss Lab, University of Washington, WA). The transitions of MRM-HR targeting each peptide were shown in Table 3. A TOF-MS scan in the mass range of 400-2,250 Da with 0.1 sec accumulation time was acquired followed by the product ion of target peptide precursors mass with 100 ms accumulation time. Collision energies were calculated with the rolling collision energy as in the information-dependent acquisition experiment. For MRM-HR data analysis, MultiQuant 3.0 Software (AB Sciex) was used to calculate integrated area of each peptide. All peak selections were checked manually after the automated matches.
2022-05-12 | PXD025947 | JPOST Repository
Project description:Human primary macrophages were infected with influenza A virus (H3N2/Udorn strain, HA 256) for 6 hrs or left untreated. Cells were collected and lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails. The cell lysates were centrifuged and the supernatant was collected and the protein content was measured with Bio-Rad DC™ protein assay (Bio-Rad). The proteins were reduced, alkylated, and enzymatically digested in-solution with trypsin. The digestion was stopped by adding FA (final c = 1 %). The samples were centrifuged 9168 x g for 10 min and desalted with Sep-Pak Vac RP C18 cartridges (Waters, MA, USA), following fractionation by strong cation exchange chromatography (SCX). The peptides were separated on a 200 x 4.6 mm, 5 μm, 200 Å PolySULFOETHYL A™ column (PolyLC, USA). The fractions were vacuum centrifuged and desalted as before, following phosphopeptide-enrichment with PHOS-Select™ Iron Affinity Gel (Sigma Aldrich, MO, USA). LC-MS/MS was performed with a Q Exactive hybrid quadrupole-orbitrap tandem mass spectrometer coupled to an EASY-nLC 1000 nanoflow liquid chromatograph (Thermo Fisher Scientific). A 100 μm x 3 cm trap column and a 75 μm x 15 cm analytical column were in-house packed with Magic C18AQ resin (200 Å, 5 μm; Michrom Bioresources). The mobile phases were 2% acetonitrile, 0.2% formic acid (A) and 95% acetonitrile, 0.2% formic acid (B). LC gradient elution condition was 2% B (0 min), 20% B (70 min), 40% B (100 min), and then 100% B (105-110 min), with a flow rate of 300 nl/min. Data dependent acquisition was performed in positive ion mode. MS spectra were acquired from m/z 300 to m/z 2000 at a resolution of 70,000 at m/z 200 with a target value of 1,000,000 and maximum injection time of 120 ms. The 10 most abundant precursor ions of which charge states were 2+ or higher were selected for higher energy collisional dissociation (HCD) with an isolation window of 2 and normalized collision energy of 30. MS/MS spectra were acquired at a resolution of 17,500 at m/z 200 with a target value of 50,000, maximum injection time of 250 ms, and the lowest mass fixed at m/z 100. Dynamic exclusion duration was 30 s.
2016-08-08 | PXD001620 | Pride
Project description:Pretreated human follicular fluid was prepared for targeted metabolomics experiments. For this purpose, 12-point standard calibration curves in the respective range with reference material were measured, and the limit of detection (LOD) and limit of quantification (LOQ) for each neurotransmitter under investigation were calculated. LC-MS/MS was conducted by an ultra-performance liquid chromatography method using a Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm, Waters, USA). The Waters acquisition UPLC was coupled to an API 4000 triple quadrupole mass spectrometer (AB SCIEX, USA). The mobile phase A buffer was 10% methanol in water (containing 0.1% formic acid), and the mobile phase B buffer was 50% methanol in water (containing 0.1% formic acid). The LC gradient elution was at a flow rate of 0.3 mL/min at 0-8.0 min and 0.4 ml/min at 8.0-17.5 min, then 10%-30% B at 0-6.5 min; 30%-100% B at 6.5-7 min; 100% B at 7-14min; and 100%-10% B at 14-17.5min. The injection volume was 5 µl and the column temperature was set to 40 °C. Twenty-three metabolites (Ach: Acetylcholine chloride; Gln: Glutamine; Glu: Glutamate; His: L-Histidine; NE: norepinephrine; Tyr:Tyrosine; Trp:Tryptophan; Kyn: Kynurenine; GABA: 4-Aminobutyric acid;HisA:Histamine; PA:Picolinic acid;TyrA:Tyramine;DA:Hydroxytyramine hydrochloride; TrpA:Tryptamine;5-HT:Serotonin hydrochloride;E:Adrenaline hydrochloride;KynA:Kynurenic acid;5-HIAA:5-Hydroxyindole-3-acetic acid;DOPA:Levodopa;XA:Xanthurenic acid;VWA:Vanillymandelic Acid; 5-HTTP:5-Hydroxytryptophan;MT:Melatonine)were simultaneously reported in LC-MS/MS multiple reaction monitoring (MRM) mode. Data analysis was performed with Analyst 1.6 software.
2022-08-12 | PXD031560 | Pride
Project description:Cells were washed with cold PBS twice and scraped from the plates, then centrifuged at 1000 rpm for 5 min to pellet cells. Washed cells were lysed with 400 L of cell lysis buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 0.15% NP-40 and proteinase inhibitor cocktail) and incubated on ice for 10 min. The suspension was carefully add at the top of sucrose buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 24% sucrose), and centrifuged at 3200g for 10 min to collect nuclear pellets. Pellets were resuspended in 250 L of Glycerol buffer (20 mM Tris-HCl pH7.5, 75 mM NaCl, 0.5 mM EDTA pH8.0, 50% glycerol), then then immediately add 250 μl Nuclear lysis buffer (10 mM Tris-HCl pH7.5, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA pH8.0, 1% NP-40, 1M urea). Mix by vortexing for 4 s and incubate on ice for 2 min. Centrifuge the lysate at 13,000 × g for 2 min to precipitate the chromatin–RNA complex. Briefly rinse the chromatin pellets with PBS-EDTA. RNA was extracted with Trizol reagent. RNA libraries were prepared according to manual of SMARTer smRNA-Seq Kit for Illumina (Clontech, 635031).
2019-10-15 | GSE134103 | GEO