Affinity-purified digested material from 5 g of leaf tissues was resuspended in 6 μL of 5% formic acid, and 5 μL injected by autosampler onto a spray tip formed from a fused silica capillary column (0.75 μm ID, 350 μm OD) generated using a laser puller. The 10 to 12 cm column was packed with C18 reversed-phase material (Reprosil-Pur 120 C18-AQ, 3 μm) by pressure bomb loading in MeOH and was pre-equilibrated with buffer A (100% H2O, 0.1% formic acid). The column was placed in-line with a LTQ-Orbitrap Elite (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (Proxeon, Thermo Fisher Scientific) connected in-line to a NanoLC-Ultra 2D plus HPLC system (Eksigent, Dublin, USA). The LTQ-Orbitrap Elite instrument under Xcalibur 2.0 was operated in the data dependent mode to automatically switch between MS and up to 10 subsequent MS/MS acquisition. The HPLC fractionation was performed on a acetonitrile gradient over 125 min. For the first twenty min, the flow rate was of 400 μL/min in 2% buffer B (100% Acetonitrile, 0.1% formic acid). The flow rate was then reduced to 200 μl/minwith a linear gradient up to 35% buffer B till 95.5 min. Buffer B was then increased to 80% over 5 min and maintained at that level until 107 min. The mobile phase was then reduced 2% buffer B until the end of the run (125 min).
Nanolc ultra 2d plus hplc system
Manufactured by AB Sciex
Sourced in United States
The NanoLC Ultra 2D Plus HPLC system is a high-performance liquid chromatography instrument designed for analytical and preparative applications. It features a dual-gradient capability, enabling the separation and analysis of complex samples with high resolution and sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Picofrit emitter, by New Objective (5 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (4 mentions)
Ltq orbitrap elite, by Thermo Fisher Scientific (2 mentions)
Magic c18 aq, by New Objective (2 mentions)
Sciex 5600 plus tripletof mass spectrometer, by AB Sciex (2 mentions)
Flag peptide, by Merck Group (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
11 protocols using nanolc ultra 2d plus hplc system
1
Affinity-Purified Leaf Peptide Analysis
2
LC-MS/MS Analysis of Phosphopeptides
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The synthetic phosphopeptide mix (without added background added) was measured on a SCIEX 5600+ TripleTOF mass spectrometer operated in DDA mode in technical triplicates. The mass spectrometer was interfaced with an Eksigent NanoLC Ultra 2D Plus HPLC system as previously described14 ,43 (link),60 (link). Peptides were directly injected onto a 20-cm PicoFrit emitter (New Objective, self-packed to 20 cm with Magic C18 AQ 3-μm 200-Å material), and then separated using a 120-min gradient from 2–35% (buffer A 0.1% (v/v) formic acid, 2% (v/v) acetonitrile, buffer B 0.1% (v/v) formic acid, 90% (v/v) acetonitrile) at a flow rate of 300 nL/min. MS1 spectra were collected in the range 360–1,460 m/z for 500 ms. The 20 most intense precursors with charge state 2–5 which exceeded 250 counts per second were selected for fragmentation, and MS2 spectra were collected in the range 50–2,000 m/z for 150 ms. The precursor ions were dynamically excluded from reselection for 20 s. Acquired file names:
3
LC-MS/MS Analysis of Phosphopeptides
4
SWATH-MS Proteomic Quantification Protocol
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Tryptic peptides were measured in SWATH mode on two TripleTOF 5600 mass spectrometers (Sciex), both interfaced with an Eksigent NanoLC Ultra 2D Plus HPLC system as described previously (Collins etal,2017 ). Peptides were separated using a 60 min gradient from 2 to 35% buffer B (0.1% (v/v) formic acid, 90% (v/v) acetonitrile). A 64‐variable window DIA scheme was applied, covering the precursor mass range of 400–1,200 m/z (for details see Extended Experimental Methods), with a total cycle time of ~ 3.45 s. Per MS injection 2 μg of protein amount was loaded onto the HPLC column. For a detailed overview of all samples measured by DIA/SWATH, see Datasets EV2 and EV3 .
5
Mass Spectrometry-Based Proteomics of iPSCs
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The peptides digested from two biological replicates of iPSC cells at time zero (cells in light medium) were both measured on an AB SCIEX 5600 plus TripleTOF mass spectrometer operated in DDA mode. The mass spectrometer was interfaced with an Eksigent NanoLC Ultra 2D Plus HPLC system as previously described [48 (link), 49 (link)]. Peptides were directly injected onto a 20-cm PicoFrit emitter (New Objective, self-packed to 20 cm with Magic C18 AQ 3-µm 200-Å material), and then separated using a 120-min linear gradient of 2 % buffer B to 35 % buffer B (buffer A 0.1 % (v/v) formic acid, 2 % (v/v) acetonitrile, buffer B 0.1 % (v/v) formic acid, 90 % (v/v) acetonitrile) at a flow rate of 300 nL/min. MS1 spectra were collected in the range 360–1,460 m/z. The 20 most intense precursors with charge state 2–5 which exceeded 250 counts per second were selected for fragmentation, and MS2 spectra were collected in the range 50–2,000 m/z for 100 ms. The precursor ions were dynamically excluded from reselection for 20 s.
6
LC-MS/MS Proteomic Analysis of E. coli
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LC‐MS/MS runs in DDA mode were performed on a TripleTOF 5600 mass spectrometer (SCIEX) interfaced with an NanoLC Ultra 2D Plus HPLC system (Eksigent). Peptides were separated using a 120 min gradient from 2 to 35% buffer B (0.1% v/v formic acid, 90% v/v acetonitrile). The 20 most intense precursors were selected for fragmentation. For the generation of the E. coli spectral library, 53 DDA‐based proteomic measurements were performed in total (see Dataset EV2 ).
7
Peptide Profiling by Data-Dependent MS
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The depleted and fractionated peptides were all measured by an AB Sciex 5600 TripleTOF mass spectrometer operated in data-dependent acquisition (DDA) mode. The mass spectrometer was interfaced with an Eksigent NanoLC Ultra 2D Plus HPLC system as previously described (Gillet et al, 2012 (link); Collins et al, 2013 (link); Liu et al, 2013b (link)). Peptides were directly injected onto a 20-cm PicoFrit emitter (New Objective, self-packed to 20 cm with Magic C18 AQ 3-μm 200-Å material) and then separated using a 120-min gradient from 2 to 35% (buffer A 0.1% (v/v) formic acid, 2% (v/v) acetonitrile, buffer B 0.1% (v/v) formic acid, 90% (v/v) acetonitrile) at a flow rate of 300 nl/min. MS1 spectra were collected in the range 360–1,460 m/z. The 20 most intense precursors with charge state 2–5 which exceeded 250 counts per second were selected for fragmentation, and MS2 spectra were collected in the range 50–2,000 m/z for 100 ms. The precursor ions were dynamically excluded from reselection for 20 s.
8
Identifying Nur77 and PEPCK1 Interactors
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For detecting the interaction protein with Nur77, Flag-Nur77 was transfected into 293T cells, and then immunoprecipitated with anti-Flag antibody and eluted with 200 μg ml−1 Flag peptide (Sigma). The proteins were separated by SDS-PAGE followed by silver staining. Visible band from silver-stained gel was cut out, and proteins were in-gel digested with trypsin, and analysed by liquid chromatography-mass spectrometry.
To detect the PEPCK1 sumoylation sites, 293T cells that transfected with Flag-PEPCK, HA-UBC9 and Myc-SUMO-1(RGG) were incubated with anti-Flag antibody. Purified PEPCK1 proteins were separated by 8% SDS-PAGE, and then stained with coomassie blue. The SUMO1-PEPCK1 conjugates were excised in separate gel slices. Proteins were digested in-gel with trypsin. The tryptic peptides were analysed on an AB Sciex TripleTOF 5600 mass spectrometer interfaced to an Eksigent NanoLC Ultra 2D Plus HPLC system. The interpretation of both the MS and MS/MS data were carried out with ProteinPilot V4.5 beta. Spectra were inspected manually to eliminate false positives, excluding spectra with low S/N, erroneous modification assignments and confidence values below 95% unless justifiable by the presence of a-ions after comparison with the theoretical MS/MS product ion spectrum.
To detect the PEPCK1 sumoylation sites, 293T cells that transfected with Flag-PEPCK, HA-UBC9 and Myc-SUMO-1(RGG) were incubated with anti-Flag antibody. Purified PEPCK1 proteins were separated by 8% SDS-PAGE, and then stained with coomassie blue. The SUMO1-PEPCK1 conjugates were excised in separate gel slices. Proteins were digested in-gel with trypsin. The tryptic peptides were analysed on an AB Sciex TripleTOF 5600 mass spectrometer interfaced to an Eksigent NanoLC Ultra 2D Plus HPLC system. The interpretation of both the MS and MS/MS data were carried out with ProteinPilot V4.5 beta. Spectra were inspected manually to eliminate false positives, excluding spectra with low S/N, erroneous modification assignments and confidence values below 95% unless justifiable by the presence of a-ions after comparison with the theoretical MS/MS product ion spectrum.
9
Mass Spectrometry-Based Proteomics of iPSCs
10
High-Resolution Mass Spectrometry of Affinity-Purified Proteins
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Affinity-purified or BioID digested material from two
150 mm plates was resuspended in 12 μL of 5% formic acid and
centrifuged at 16,100g for 1 min before 5 μL
was injected by autosampler to a homemade high-performance liquid
chromatography (HPLC) column (75 μm ID, 360 μm OD with
spray tip generated using a laser puller) loaded with 10–12
cm of C18 reversed-phase material (ReproSil-Pur 120 C18-AQ 3 mm).
The column was placed in line with an LTQ-Orbitrap Elite (Thermo Fisher
Scientific) equipped with a nanoelectrospray ion source connected
in line to a NanoLC-Ultra 2D plus HPLC system (Eksigent, Dublin).
The LTQ-Orbitrap Elite instrument under Xcalibur 2.0 was operated
in the data-dependent acquisition mode to automatically switch between
one MS1 survey scan and up to 10 MS2 scans. MS1 scans at 60k res with
1 × 106 target and MS2 used CID fragmentation and
detection in ion trap with 3 × 104 target. Dynamic
exclusion of 15 s. Buffer A is 99.9% H2O, 0.1% formic acid;
buffer B is 99.9% acetonitrile, 0.1% formic acid. Peptides eluted
over 90 min from 2 to 35% acetonitrile with a flow rate of 200 nL/min.
150 mm plates was resuspended in 12 μL of 5% formic acid and
centrifuged at 16,100g for 1 min before 5 μL
was injected by autosampler to a homemade high-performance liquid
chromatography (HPLC) column (75 μm ID, 360 μm OD with
spray tip generated using a laser puller) loaded with 10–12
cm of C18 reversed-phase material (ReproSil-Pur 120 C18-AQ 3 mm).
The column was placed in line with an LTQ-Orbitrap Elite (Thermo Fisher
Scientific) equipped with a nanoelectrospray ion source connected
in line to a NanoLC-Ultra 2D plus HPLC system (Eksigent, Dublin).
The LTQ-Orbitrap Elite instrument under Xcalibur 2.0 was operated
in the data-dependent acquisition mode to automatically switch between
one MS1 survey scan and up to 10 MS2 scans. MS1 scans at 60k res with
1 × 106 target and MS2 used CID fragmentation and
detection in ion trap with 3 × 104 target. Dynamic
exclusion of 15 s. Buffer A is 99.9% H2O, 0.1% formic acid;
buffer B is 99.9% acetonitrile, 0.1% formic acid. Peptides eluted
over 90 min from 2 to 35% acetonitrile with a flow rate of 200 nL/min.
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