Lysates of NCI-H23 cells infected with control lentivirus or overexpressing MAL2 were further lysed with sonication at 4 °C for 3 min (80 W, on 1 s and off 1 s) and centrifuged at 12,000×g at 4 °C for 10 min to remove insoluble particles, then centrifuged one more time and the supernatant was collected. The samples were fractionated using sequencing-grade trypsin in 100 mM TEAB buffer (Sigma). Then, the samples were labeled using the TMT label reagent (Thermo scientific) according to the manufacturer’s instructions. Phosphorylated peptides were enriched by using titanium dioxide beads (TiO2). Mass spectrometry analysis was performed by Shanghai OE-biotech (China) with an EASY-nLCTM 1200 system (Thermo, USA) in Q-Exactive mass spectrometer equipped with a Nanospray Flex source (Thermo, USA). The data were processed with Proteome Discoverer™ 2.2 (Thermo, USA) software against the Homo sapiens Uniprot database. The phosphorylatedproteins with an average fold change (FC > 1.2; P < 0.05) in the experimentally treated groups were considered to be the differentially accumulated proteins.
Nanospray flex source
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nanospray Flex source is a compact, high-sensitivity ion source designed for nanoscale liquid chromatography-mass spectrometry (nanoLC-MS) applications. It provides efficient ionization and ion transfer to the mass spectrometer, enabling the analysis of low-level analytes in complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive mass spectrometer, by Thermo Fisher Scientific (13 mentions)
Easy nlctm 1200 system, by Thermo Fisher Scientific (5 mentions)
Chromxp eksigent system, by AB Sciex (4 mentions)
Zorbax extend rp column, by Agilent Technologies (4 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (3 mentions)
1100 hplc system, by Agilent Technologies (3 mentions)
Acclaim pepmap rslc, by Thermo Fisher Scientific (2 mentions)
Easy nlc, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 rslc nano uhplc, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion trihybrid, by Thermo Fisher Scientific (2 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (1 mentions)
Teab buffer, by Merck Group (1 mentions)
Tmt label reagent, by Thermo Fisher Scientific (1 mentions)
Q exactive ms, by Thermo Fisher Scientific (1 mentions)
Xcalibur v2, by Thermo Fisher Scientific (1 mentions)
Orbitrap elite etd mass spectrometer, by Thermo Fisher Scientific (1 mentions)
40mm stainless steel emitter, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000 uhplc system, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap100, by Thermo Fisher Scientific (1 mentions)
24 protocols using nanospray flex source
1
Phosphoproteome Analysis of MAL2 Overexpression
2
Mass Spectrometric Analysis of Peptide Mixtures
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The MS analysis was performed on a Q-Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a Nanospray Flex source (Thermo Fisher Scientific). The peptide mixtures were loaded by a capillary C18 trap column (3 cm × 100 μm, C18, 3 μm, 150 Å) and separated by a C18 column (15 cm × 75 μm, C18, 3 μm, 120 Å) on an ChromXP Eksigent system (ABSciex). The flow rate was 300 nl/min and the linear gradient was 70 min (0–0.5 min, 95–92% A; 0.5–48 min, 92–74% A; 48–61 min, 74–62% A; 61–61.1 min, 62–15% A; 61.1–67 min, 15% A; 67–67.1, 15–95% A; 67.1–70 min, 95% A. Mobile phase A = 2% acetonitrile (ACN)/0.1% formic acid (FA) and B = 95% ACN/0.1% FA. Full MS scans were acquired in the mass range of 300–1600 m/z with a mass resolution of 70 000, and the AGC target value was set at 1e6. The 10 most intense peaks in MS were fragmented with higher energy collisional dissociation with a collision energy of 30. MS/MS spectra were obtained with a resolution of 17 500 with an AGC target of 2000 00 and a maximum injection time of 50 ms. The QE dynamic exclusion was set for 15.0 s and run under positive mode. The MS data were processed and analyzed with the help of Oebiotech Co., Ltd (Shanghai, China). The raw data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org ) via the iProX partner repository with the dataset identifier PXD032864.
3
Nano-LC-MS/MS Proteomic Analysis
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All analyses were performed using a Q-Exactive mass spectrometer (Thermo Fisher, Waltham, MA, USA) equipped with a Nanospray Flex source (Thermo, Waltham, MA, USA). The samples were loaded at a flow rate of 2 µL/min onto a pre-column, Acclaim PepMap100 100 μm × 2 cm (RP-C18, Thermo Fisher, Waltham, MA, USA) (loading time 3 min), and then they were separated at a flow rate of 300nL/min onto an analytical column, Acclaim PepMap RSLC, 75 μm × 50 cm (RP-C18, Thermo Fisher, Waltham, MA, USA). The linear gradient was 75 min (0~40 min, 5–28% B; 40~60 min, 28–42% B; 60~65 min, 42–90% B; 60~65 min, 90% B; mobile phase A = 0.1% formic acid (FA, Thermo Fisher, Waltham, MA, USA) in water and B = 0.1% FA in ACN). The first MS resolution was set to 60,000, the automatic gain control value was set to 1 × 106, and the maximum injection time was 50 ms. The mass spectral scan was set to a full-scan charge-to-mass ratio m/z range of 350–1500; all MS/MS plot acquisitions were accomplished using high-energy collisional cleavage in the data-dependent positive-ion mode, with the collisional energy set to 36. The MS/MS resolution was set to 30,000, the automatic gain control was set to 1 × 105, and the maximum accumulation time of ions was set to 80 ms; the dynamic exclusion time was set to 30 s.
4
LC-MS/MS Analysis of Peptide Mixtures
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All LC-MS/MS analyses were performed on a Q-Exactive mass spectrometer (Thermo, USA) equipped with a Nanospray Flex source (Thermo, USA). The peptides mixtures were loaded by a capillary C18 trap column (3 cm × 100 µm, C18, 3 µm, 150 Å) and separated by a C18 column (15 cm × 75 µm, C18, 3 µm, 120 Å) on an ChromXP Eksigent system (AB Sciex). The flow rate was 300 nL/min and linear gradient was 70 min (0–0.5 min, 95–92% A; 0.5–48 min, 92–74% A; 48–61 min, 74–62% A; 61–61.1 min, 62–15% A; 61.1–67 min, 15% A; 67–67.1, 15–95% A; 67.1–70 min, 95% A. Mobile phase A = 2% ACN/0.1% FA and B = 95% ACN/0.1% FA). Full MS scans were acquired in the mass range of 300–1600 m/z with a mass resolution of 70000 and the AGC target value was set at 1e6. The ten most intense peaks in MS were fragmented with higher-energy collisional dissociation with collision energy of 30. MS/MS spectra were obtained with a resolution of 17500 with an AGC target of 200000 and a max injection time of 50 ms. The Q-E dynamic exclusion was set for 15.0 s and run under positive mode. The MS data were performed and analyzed with the help of the commercial biotechnology company Oebiotech (Shanghai, China).
5
Quantitative Proteomics Analysis Using Q-Exactive MS
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Compared with MS, LC–MS analyses were performed on a Q-Exactive mass spectrometer (Thermo, USA) equipped with a Nanospray Flex source (Thermo, USA). The peptides mixtures were loaded by a capillary C18 trap column (3 cm × 100 μm, C18, 3 μm, 150 A) and separated by a C18 column (15 cm × 75 μm, C18, 3 μm, 120 A) on a ChromXP Eksigent system (AB Sciex). Full MS scans were acquired in the mass range of 300–1600 mass compared with a mass resolution of 70,000, and the AGC target value was set at 1,000,000. The 10 most intense peaks in MS were fragmented with higher-energy collisional dissociation (HCD) with a collision energy of 30%. MS spectra were obtained with a resolution of 17,500 with an AGC target of 200,000 and a max injection time of 50 ms. The Q-E dynamic exclusion was set for 15.0 s and run under positive mode61 .
6
Phosphorylated Peptide Analysis by Q-Exactive
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The enriched phosphorylated peptides were analyzed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a Nanospray Flex source (Thermo Fisher Scientific). The samples were loaded and separated by a C18 column on an EASY-nLCTM 1200 system (Thermo Fisher Scientific). The full scan was performed in the mass range of 300–1,500 m/z with a mass resolution of 70,000. The 10 most intense peaks in MS were fragmented with higher-energy collisional dissociation with normalized collisional energy (NCE) of 32. MS/MS spectra were obtained with a resolution of 17,500 with a maximum injection time of 80 ms. The Q-E dynamic exclusion was set for 30 s and run under positive mode.
7
HPLC-based Proteomics Analysis of Mycobacterium tuberculosis
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A 1100 HPLC system (Agilent) was used for reversed-phase (RP) chromatographic separation with an Agilent Zorbax Extend RP column (5 µm, 150 × 2.1 mm). Mobile phases A (2% acetonitrile in HPLC water) and B (98% acetonitrile in HPLC water) were used for the RP gradient. Trypsin peptides were separated at a steady flow rate of 300 µl/min, monitored at 210 and 280 nm. Then elution buffer at every minute was collected, lasting for 8–50 min. The separated peptides were lyophilized for mass spectrometer (MS) detection as follows.
All analyses were performed by a Q-Exactive MS (Thermo, United States) equipped with a nanospray flex source (Thermo, United States). The peptides were separated on a C18 analytical RP column (75 µm × 15 cm) at a flow rate of 300 nL/min and a linear gradient of 70 min. A full MS scan was obtained in the mass range of 300 to 1600 m/z. Ten strongest peaks in the MS were fragmented by high-energy collision dissociation. The resolution of the obtained MS/MS spectrum is 17,500 with the maximum injection time of 50 ms. Q-E dynamic exclusion was set to 15 s.
All of the Q exactive MS/MS raw data were analyzed by using proteome discoverer v.2.2 (Thermo Company, United States) software and the uniprot-M.tb database. The false-positive rate of peptide identification was controlled below 1%.
All analyses were performed by a Q-Exactive MS (Thermo, United States) equipped with a nanospray flex source (Thermo, United States). The peptides were separated on a C18 analytical RP column (75 µm × 15 cm) at a flow rate of 300 nL/min and a linear gradient of 70 min. A full MS scan was obtained in the mass range of 300 to 1600 m/z. Ten strongest peaks in the MS were fragmented by high-energy collision dissociation. The resolution of the obtained MS/MS spectrum is 17,500 with the maximum injection time of 50 ms. Q-E dynamic exclusion was set to 15 s.
All of the Q exactive MS/MS raw data were analyzed by using proteome discoverer v.2.2 (Thermo Company, United States) software and the uniprot-M.tb database. The false-positive rate of peptide identification was controlled below 1%.
8
Shotgun Proteomic Analysis by LC-MS/MS
9
Peptide Characterization by Q-Exactive HF
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All analyses were performed by a Q-Exactive HF mass spectrometer (Thermo) equipped with a Nanospray Flex source (Thermo). Samples were loaded and separated by a C18 column (15 cm × 75 µm) on an EASY-nLCTM 1200 system (Thermo). The flow rate was 300 nL/min and linear gradient was 60 min (0–1 min, 2–9 %B; 1–45 min, 9–29 % B; 45–52 min, 29–37 % B; 52–56 min, 37–100 % B; 56–60 min, 100 %B; mobile phase A = 0.1 % FA in water and B = 0.1 % FA in ACN). Full MS scans were acquired in the mass range of 350–1500 m/z with a mass resolution of 60,000, and the AGC target value was set at 3e6. The 10 most intense peaks in MS were fragmented with higher-energy collisional dissociation (HCD) at a collision energy of 32 MS/MS. Spectra were obtained with a resolution of 45,000 with an AGC target of 2e5 and a maximum injection time of 80 ms. The Q-Exactive HF dynamic exclusion was set for 30 s and run under positive mode.
10
Q-Exactive LC-MS/MS Peptide Analysis
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All LC-MS/MS analyses were performed on a Q-Exactive mass spectrometer (Thermo, USA) equipped with a Nanospray Flex source (Thermo, USA). The peptides mixtures were loaded by a capillary C18 trap column (3 cm × 100 μm, C18, 3 μm, 150 Å) and separated by a C18 column (15 cm × 75 μm, C18, 3 μm, 120 Å) on an ChromXP Eksigent system (AB Sciex). The flow rate was 300 nL·min− 1 and linear gradient was 70 min (0~0.5 min, 95%~ 92% A; 0.5~48 min, 92%~ 74% A; 48~61 min, 74%~ 62% A; 61~61.1 min, 62%~ 15% A; 61.1~67 min, 15% A; 67~67.1, 15%~ 95% A; 67.1~70 min, 95% A. mobile phase A = 2% ACN/0.1% FA and B = 95% ACN/0.1% FA). Full MS scans were acquired in the mass range of 300–1600 m/z with a mass resolution of 70,000 and the AGC target value was set at 1,000,000. The 10 most intense peaks in MS were fragmented with higher-energy collisional dissociation (HCD) with collision energy of 30. MS/MS spectra were obtained with a resolution of 17,500 with an AGC target of 200,000 and a max injection time of 50 ms. The Q-E dynamic exclusion was set for 15.0 s and run under positive mode.
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