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.
Easy nlctm 1200 system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EASY-nLCTM 1200 system is a nano-flow liquid chromatography (nano-LC) instrument designed for high-performance separations of complex biological samples. It features a compact and robust design to provide reliable, reproducible, and high-resolution chromatographic separations for a wide range of applications, including proteomics, metabolomics, and lipidomics.
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Lab products found in correlation
Nanospray flex source, by Thermo Fisher Scientific (5 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (5 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Nanospray flex, by Thermo Fisher Scientific (2 mentions)
Rp c18, by Thermo Fisher Scientific (2 mentions)
Easy spray source, by Thermo Fisher Scientific (2 mentions)
Qe mass spectrometer, 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)
Acclaim pepmap100, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap rslc, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap 100 pre column, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive plus orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer, by Thermo Fisher Scientific (1 mentions)
Q exactive hf x hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
14 protocols using easy nlctm 1200 system
1
Phosphoproteome Analysis of MAL2 Overexpression
2
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.
3
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.
4
Proteome Analysis by Q-Exactive Mass Spectrometry
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All analyses were performed by a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA) coupled with a Nanospray Flex source (Thermo Fisher Scientific, USA) as described earlier [7 (link)]. Samples were redissolved with 0.1% formic acid and separated by a C18 column (15 cm × 75 µm) on an EASY-nLCTM 1200 system (Thermo Fisher Scientific, USA). The flow rate was 300 nL/min, and the linear gradient was 90 min. Full MS scans were acquired in the mass range of 300–1,600 m/z with a mass resolution of 70,000, and the automatic gain control (AGC) target value was set at 1e6. The ten most intense peaks in MS were fragmented with higher energy collisional dissociation with positive polarity in the data-dependent mode. MS/MS spectra were obtained with a resolution of 17,500 with an AGC target of 2e5 and a max injection time of 80 ms. Fragmentation was performed with normalized collision energy of 32 and dynamic exclusion of 30 s.
5
Phosphopeptide Characterization by LC-MS/MS
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The enriched phosphopeptide samples were analyzed by LC-MS/MS on a Nanospray Flex source coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA). Peptide mixtures were firstly loaded onto a C18 nano-trap column (Acclaim PepMap100, 200 mm length × 100 μm inside diameter, Thermo Fisher Scientific) and then separated on an analytical column (Acclaim PepMap RSLC, 150 mm length × 75 μm inside diameter) on an EASY-nLCTM 1200 system (Thermo Fisher Scientific). The mobile phases consisted of 0.1% formic acid (A) and 0.1% formic acid and 80% ACN (B) in a three-step linear gradient of 0–20% solution B over 47 min, 20–40% solution B over 26 min, and 40–90% solution B over 4 min.
In positive ion mode, the mass spectrometry full scan range had a mass/charge ratio (m/z) of 300-1600 with a mass resolution of 70,000, and the twelve most intense peaks in MS were selected and further fragmented with higher-energy collisional dissociation with a normalized collision energy of 30. MS/MS spectra were acquired at 17500 resolution with a max injection time of 80 ms, and the dynamic exclusion time was set to 30 s.
In positive ion mode, the mass spectrometry full scan range had a mass/charge ratio (m/z) of 300-1600 with a mass resolution of 70,000, and the twelve most intense peaks in MS were selected and further fragmented with higher-energy collisional dissociation with a normalized collision energy of 30. MS/MS spectra were acquired at 17500 resolution with a max injection time of 80 ms, and the dynamic exclusion time was set to 30 s.
6
Glycopeptide Analysis by Orbitrap Fusion Lumos
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The samples of TMT-labeled peptide and intact glycopeptide were separated in an Easy-nLCTM 1200 system (Thermo Fisher Scientific) by using Acclaim PepMap100 precolumn (2 cm, 75 μm i.d., 3 μm) and Acclaim Pepmap100 separation column (50 cm, 75 μm i.d., 3 μm). The mobile-phase flow rate was 200 nL/min and consisted of 0.1% formic acid in water (A) and 0.1% formic acid in 80% acetonitrile (B). For the analysis with five fragmentation energies, the gradient profile was set as follows: 3–7% B for 3 min, 7–35% B for 91 min, 35–68% B for 19 min, 68–99% B for 4 min, and equilibrated in 100% B for 13 min. MS analysis was performed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Germany). The spray voltage was set at 2400 V. Orbitrap spectra (AGC 4×105) were collected from 350 m/z to 2000 m/z at a resolution of 120 K followed by oxonium ions triggered by data-dependent higher-energy collisional dissociation (HCD) MS/MS (at a resolution of 50 K, collision energy 20%, AGC 5×104, and collision energy 37%, AGC 2×105) using an isolation width of 1.6 Da. Charge state screening (2–7) was enabled to reject unassigned and singly charged ions. Polypeptides with a scanning range of 110–3000 m/z were selected for MS/MS collection. A dynamic exclusion time of 5 s was used to discriminate against previously selected ions.22 (link)
7
Proteomic Analysis of Frozen Leaf Samples
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Frozen leaf samples (approximately 1.0 g for each replicate) were ground to a fine powder using liquid nitrogen, and were then lysed ultrasonically. An equal volume of Tris-phenol was added, and the samples were centrifuged at 12,000× g for 10 min at 4 °C. Proteins were precipitated, washed with methanol and acetone, and the final pellet was resuspended in 1% SDS. Protein concentrations were determined and then digested according to a previously described procedure [18 (link)]. LC-MS was performed using the Q Exactive TM Plus Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Rockford, IL, USA) and the Easy-nLCTM 1200 system (Thermo Fisher Scientific). Parameters were set as follows: electrospray voltage, 2.1 kV; automatic gain control (AGC), 5E4; survey scans were acquired at a resolution of 1,200,000; resolution for HCD spectra, 15,000.
8
Quantitative Proteomics Analysis Using Q-Exactive HF
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This work adopted the Q-Exactive HF mass spectrometer (Thermo, USA) for MS analysis through using the Nanospray Flex source (Thermo, USA). Furthermore, the C18 column (15 cm × 75 μm) was utilized to load and separate samples using the EASY-nLCTM 1200 system (Thermo, USA). We set the linear gradient and flow rate at 75 min (5%–45% B in 0–63 min, 45%–90% B in 63–65 min, 90% B in 65–67 min; mobile phase A consisted of 0.1% FA contained within water, whereas mobile phase B included 0.1% FA supplemented within ACN) and 300 nl/min, separately.
This work later obtained full MS scans within the 350–1500 m/z mass range with the AGC target value and mass resolution being 3e6 and 60,000, respectively. From MS spectra, we fragmented 20 peaks with the strongest intensity through higher-energy collisional dissociation (HCD), and the collision energy was 30. The following parameters were used to obtain MS/MS spectra: resolution = 15,000, max injection time = 40 ms, and AGC target = 2e5. Using the positive mode, this work set Q Exactive HF dynamic exclusion at 30.0 s.
This work later obtained full MS scans within the 350–1500 m/z mass range with the AGC target value and mass resolution being 3e6 and 60,000, respectively. From MS spectra, we fragmented 20 peaks with the strongest intensity through higher-energy collisional dissociation (HCD), and the collision energy was 30. The following parameters were used to obtain MS/MS spectra: resolution = 15,000, max injection time = 40 ms, and AGC target = 2e5. Using the positive mode, this work set Q Exactive HF dynamic exclusion at 30.0 s.
9
4D-DIA Mass Spectrometry Analysis
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4D-DIA mass spectrometry (MS) analysis was performed as the samples were separated using a C18 column (25 cm × 75 µm) on an EASY-nLCTM 1200 system (Thermo Fisher, Waltham, MA, USA)60 (link),61 (link). The flow rate was 300 nL/min and the linear gradient was set accordingly. The DDA MS data for the library was acquired via the PASEF method as follows: MS data were collected over an m/z range of 100–1700, and during each MS/MS data collection, each TIMS cycle time was 1.1 s; each cycle included 1 MS and 10 MS/MS 100 msec TIMS scans; in each of the 10 PASEF MS/MS scans an average of 12 precursors were selected, resulting in an MS/MS data acquisition rate of 109 Hz. For the DIA, 56 DIA windows were acquired (automatic gain control target 3e6 and auto for injection time), and the collision energy was ramped linearly as a mobility function from 59 eV at 1/K0 = 1.6 Vs cm−2 to 20 eV at 1/K0 = 0.6 Vs cm−2. The MS/MS spectra were recorded from 100 to 1700 m/z.
10
Mass Spectrometry-Based Proteomic Analysis
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All analyses were performed by a Q-Exactive mass spectrometer (Thermo, USA) equipped with a Nanospray Flex source (Thermo, USA). Samples were loaded and separated by a C18 column (15 cm × 75 μm) on an EASY-nLCTM 1200 system (Thermo, USA). Full MS scans were acquired in the mass range of 350-1650 m/z with a mass resolution of 120,000 with an AGC target of 3e6. MS/MS spectra were obtained with a resolution of 60,000 with an AGC target of 1e5 and a max injection time of 120 ms. The Q-E dynamic exclusion was set for 40.0 s and run under positive mode. Proteome Discoverer (v.2.4) was used to search all of the Q Exactive raw data. Database search was performed with trypsin digestion specificity. A global false discovery rate (FDR) was set to 0.01 and protein groups considered for quantification required at least 2 peptides.
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