A pool of mouse liver ADPr peptides from the pilot study were subjected to a dual column setup: an Acclaim PepMap RSLC C18 trap column, 75 μm 20 mm (Thermo Fisher Scientific, Cat# 164261); and an EASY-Spray LC Column, 75 μm × 250 mm (Thermo Fisher Scientific, Cat# ES802A). The analytical gradient for the ADPr peptide pool was run at 300 nl/min from 5 to 21 % Solvent B (acetonitrile/0.1% formic acid) for 50 min, followed by 10 min of 21 to 30% Solvent B, and another 10 min of a jigsaw wash (alternating between 5 and 95% Solvent B) to clean the column. Solvent A was water/0.1% formic acid. The instrument was set to 120 K resolution, and the top N precursor ions (within a scan range of m/z 400–1500) in 3 s cycle time were subjected to MS/MS. Dynamic exclusion was enabled (60 s), the isolation width was m/z 1.2, and the resolution was 120 K (automatic gain control, AGC, 1.0e4). HCD collision energies were set to 20%, 24%, 26%, 28%, 30%, 32%, or 34%. The CID collision energy settings were 20%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, and 40%.
Easy spray lc column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EASY-Spray LC column is a liquid chromatography column designed for high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) applications. The column features a pre-packed stationary phase that enables efficient separation and analysis of a variety of chemical compounds.
Automatically generated - may contain errors
Lab products found in correlation
Acclaim pepmap rslc c18 trap column, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion lumos, by Thermo Fisher Scientific (2 mentions)
Easy nlc1000 hplc pump, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
Orbitrap elite, by Thermo Fisher Scientific (2 mentions)
Easy spray source, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion lumos tribrid, by Thermo Fisher Scientific (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Q exactive hf hybrid quadrupole orbitrap ms, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 rslcnano system, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200, by Thermo Fisher Scientific (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
11 protocols using easy spray lc column
1
Dual Column Workflow for ADP-Ribosylated Peptide Analysis
2
VIC Peptide Analysis by Orbitrap Fusion
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VIC peptide samples were analyzed on the Orbitrap Fusion Lumos mass spectrometer fronted with an EASY-Spray Source (heated at 45 °C) and coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific). Peptides were subjected to a dual-column setup: an Acclaim PepMap RSLC C18 trap analytical column, 75 μm × 20 mm (precolumn) and an EASY-Spray LC column, 75 μm × 250 mm (Thermo Fisher Scientific). The analytical gradient was run at 300 nl/min from 5 to 21% solvent B (acetonitrile/0.1% formic acid) for 75 min, 21 to 30% solvent B for 15 min, followed by 10 min of 95% solvent B. Solvent A was water/0.1% formic acid. The acetonitrile and water were of MS grade. The Orbitrap analyzer was set to 120 K resolution, and the top N precursor ions in 3 s cycle time within a scan range of 375 to 1500 m/z (60 s dynamic exclusion enabled) were subjected to collision-induced dissociation (collision energy, 30%; isolation window, 1.6 m/z; automatic gain control target, 1.0 e4). The ion trap analyzer was set to a rapid scan rate for peptide sequencing (MS/MS).
3
Orbitrap-based Proteomics Workflow
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All samples were analyzed
on a linear iontrap–orbitrap
mass spectrometer (Orbitrap Elite, Thermo Fisher Scientific, Rockford,
IL) coupled online to a liquid chromatograph (Ultimate 3000 RSLCnano
Systems, Dionex, Thermo Fisher Scientific, UK) with a C18-column (EASY-Spray
LC Column, Thermo Fisher Scientific, Rockford, IL). The flow rate
was 0.2 μL/min using 98% mobile phase A (0.1% formic acid) and
2% mobile phase B (80% acetonitrile in 0.1% formic acid). To elute
the peptides, the percentage of mobile phase B was first increased
to 40% over a time course of 110 min followed by a linear increase
to 95% in 11 min. Full MS scans were recorded in the orbitrap at a
120,000 resolution for MS1 with a scan range of 300–1700 m/z. The 20 most intense ions (precursor
charge ≥2) were selected for fragmentation by collision-induced
disassociation, and MS2 spectra were recorded in the ion trap (20,000
ions as a minimal required signal, 35 normalized collision energy,
dynamic exclusion for 40 s).
on a linear iontrap–orbitrap
mass spectrometer (Orbitrap Elite, Thermo Fisher Scientific, Rockford,
IL) coupled online to a liquid chromatograph (Ultimate 3000 RSLCnano
Systems, Dionex, Thermo Fisher Scientific, UK) with a C18-column (EASY-Spray
LC Column, Thermo Fisher Scientific, Rockford, IL). The flow rate
was 0.2 μL/min using 98% mobile phase A (0.1% formic acid) and
2% mobile phase B (80% acetonitrile in 0.1% formic acid). To elute
the peptides, the percentage of mobile phase B was first increased
to 40% over a time course of 110 min followed by a linear increase
to 95% in 11 min. Full MS scans were recorded in the orbitrap at a
120,000 resolution for MS1 with a scan range of 300–1700 m/z. The 20 most intense ions (precursor
charge ≥2) were selected for fragmentation by collision-induced
disassociation, and MS2 spectra were recorded in the ion trap (20,000
ions as a minimal required signal, 35 normalized collision energy,
dynamic exclusion for 40 s).
4
Orbitrap Mass Spectrometry Analysis of Protein Complexes
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TAF4–TAF12 complexes
were analyzed
on an Orbitrap Fusion Lumos Tribrid (Thermo Fisher Scientific) and
yeast lysates were analyzed on an Orbitrap Elite (Thermo Fisher Scientific).
Both were coupled online to an Ultimate 3000 RSLCnano Systems (Dionex,
Thermo Fisher Scientific). Peptides were loaded onto an EASY-Spray
LC Column (Thermo Fisher Scientific) at a flow rate of 0.300 μL
min–1 using 98% mobile phase A (0.1% formic acid)
and 2% mobile phase B (80% acetonitrile in 0.1% formic acid). To elute
the peptides, the percentage of mobile phase B was first increased
to 40% over a time course of 110 min followed by a linear increase
to 95% in 11 min.
Full MS scans for yeast lysates were recorded
in the orbitrap at 120 000 resolution for MS1 with a scan range
of 300–1700 m/z. The 20 most
intense ions (precursor charge ≥2) were selected for fragmentation
by collision-induced disassociation, and MS2 spectra were recorded
in the ion trap (2.0 × 104 ions as a minimal required
signal, 35 normalized collision energy, dynamic exclusion for 40 s).
Both MS1 and MS2 were recorded in the orbitrap for the TAF4–TAF12
complexes (120 000 mass resolution for MS1 and 15 000
mass resolution for MS2, scan range 300–1700 m/z, dynamic exclusion for 60 s, precursor charge
≥3). Higher-energy collision dissociation was used to fragment
peptides (30% collision energy, 5.0 × 104 AGC target,
60 ms maximum injection time).
were analyzed
on an Orbitrap Fusion Lumos Tribrid (Thermo Fisher Scientific) and
yeast lysates were analyzed on an Orbitrap Elite (Thermo Fisher Scientific).
Both were coupled online to an Ultimate 3000 RSLCnano Systems (Dionex,
Thermo Fisher Scientific). Peptides were loaded onto an EASY-Spray
LC Column (Thermo Fisher Scientific) at a flow rate of 0.300 μL
min–1 using 98% mobile phase A (0.1% formic acid)
and 2% mobile phase B (80% acetonitrile in 0.1% formic acid). To elute
the peptides, the percentage of mobile phase B was first increased
to 40% over a time course of 110 min followed by a linear increase
to 95% in 11 min.
Full MS scans for yeast lysates were recorded
in the orbitrap at 120 000 resolution for MS1 with a scan range
of 300–1700 m/z. The 20 most
intense ions (precursor charge ≥2) were selected for fragmentation
by collision-induced disassociation, and MS2 spectra were recorded
in the ion trap (2.0 × 104 ions as a minimal required
signal, 35 normalized collision energy, dynamic exclusion for 40 s).
Both MS1 and MS2 were recorded in the orbitrap for the TAF4–TAF12
complexes (120 000 mass resolution for MS1 and 15 000
mass resolution for MS2, scan range 300–1700 m/z, dynamic exclusion for 60 s, precursor charge
≥3). Higher-energy collision dissociation was used to fragment
peptides (30% collision energy, 5.0 × 104 AGC target,
60 ms maximum injection time).
5
MS-based Protein Identification and Quantification
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For a detailed description of MS and data processing, see Text S1 in the supplemental material. Analysis was performed with an EASY-Spray LC column coupled to a Q Exactive mass spectrometer via an EASY-Spray Source (Thermo Fischer Scientific) in a data-dependent acquisition mode (data were processed using MaxQuant version 1.5.7.4).
6
Orbitrap Fusion Lumos Mass Spectrometry Protocol
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All peptide samples were analyzed
on an Orbitrap Fusion Lumos mass spectrometer fronted with an EASY-Spray
Source, coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific).
The peptides were subjected to a dual column setup: an Acclaim PepMap
RSLC C18 trap column, 75 μm × 20 mm (Thermo Fisher Scientific,
Cat# 164261); and an EASY-Spray LC Column, 75 μm × 250
mm (Thermo Fisher Scientific, Cat# ES802). The analytical gradient
was run at 300 nL/min from 5 to 21% Solvent B (acetonitrile/0.1% formic
acid) for 50 min, 21 to 30% Solvent B for 10 min, and 95% Solvent
B for 5 min. Solvent A was water/0.1% formic acid.
on an Orbitrap Fusion Lumos mass spectrometer fronted with an EASY-Spray
Source, coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific).
The peptides were subjected to a dual column setup: an Acclaim PepMap
RSLC C18 trap column, 75 μm × 20 mm (Thermo Fisher Scientific,
Cat# 164261); and an EASY-Spray LC Column, 75 μm × 250
mm (Thermo Fisher Scientific, Cat# ES802). The analytical gradient
was run at 300 nL/min from 5 to 21% Solvent B (acetonitrile/0.1% formic
acid) for 50 min, 21 to 30% Solvent B for 10 min, and 95% Solvent
B for 5 min. Solvent A was water/0.1% formic acid.
7
Mass Spectrometry Proteomics Workflow
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All solvents were LCMS grade purchased from Fischer Scientific (Hampton, NH, USA). Peptides from each fraction were reconstituted with 15 μL mobile phase A (water/0.1% formic acid) and a 6 μL injection volume was drawn by Dionex Ultimate 3000 RSLCnano system (Thermo Scientific, Waltham, MA, USA) with C18 column (EASY-Spray LC column, 2 μm particle size, 75 μm D × 25 cm L; Thermo Scientific) heated at 55 °C. Gradient for nano/cap pump at a flow rate of 0.3 μL/min was set as followed: 5% mobile phase B (ACN/0.1% formic acid) at 0 min, 7% at 5 min, 25% at 90 min, 60% at 108 min, 95% at 113–123 min, 2% at 125–135 min. Peptides were ionized by electrospray ionization (EASY-Spray Source; Thermo Scientific) and analyzed by Q Exactive HF hybrid quadrupole-Orbitrap MS (Thermo Scientific). MS was operated at positive polarity with full MS scan (resolution: 120,000, 350–1800 m/z) followed by data dependent MS2 scan (resolution: 15,000, normalized collision energy: 28, isolated top 20 most intense ions).
8
Nano-LC-MS/MS Peptide Identification
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Peptide material from pull-down experiments was resuspended in 20 μl water with 0.1% TFA and 2% acetonitrile. Samples were then injected into a Dionex Ultimate 3000 nano-ultra LC system (Thermo Fisher) coupled on-line to a Q Exactive mass spectrometer (Thermo Scientific) as described previously (27 (link), 28 (link)). In brief, the samples were separated on an EASY-Spray LC column (PepMap RSLC C18, 500 mm × 75 μm ID, 2 μm particle size, Thermo Scientific) over 60 min for GlyGly immunoprecipitation and 120 min for the total proteome (gradient of 2–35% acetonitrile in 5% DMSO and 0.1% formic acid) at 250 nl/min. Acquisition of MS1 scans was performed at a resolution of 60,000 at 200 m/z. The most abundant precursor ions (top 12) were selected for HCD fragmentation.
9
Mass Spectrometry of Peptide Separation
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Peptides were separated using a 50 cm EASY-Spray™ LC column (Thermo Scientific) and the EASY-LC 1000 chromatography system (Thermo Scientific). Separation was achieved by a 120 min linear gradient from 2 to 26% acetonitrile in 0.1% formic acid at a flow rate of 300 nL/min. Eluting peptides were ionized by electron spray and analyzed by an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). The survey MS spectrum was acquired at a resolution of R = 120,000 in the range of m/z 200–2000. MS/MS data for the 20 most intense precursors were obtained using higher-energy collisional dissociation for ions with a charge z > 1 at a resolution of R = 15,000.
10
High-Resolution Proteome Profiling
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Each sample was divided into two technical replicates. The desalted samples were reconstituted with 3% acetonitrile in 0.1% formic acid, and a pooled sample of all samples was prepared to serve as an alignment reference in the quantification analyses stage. Randomization for sample run order was applied and the samples were individually analyzed in a Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific), coupled with EASY-Spray™ LC Columns (Thermo Scientific) column and emitter for chromatographic separation, and a linear gradient of acetonitrile/water (2 to 35% acetonitrile in 80 min, containing 0.1% formic acid) with a flow rate of 300 nl/min, and an automatic switching between MS and MS/MS scans using a top-12 method. MS spectra were acquired for a mass range of 300–1700 m/z in profile mode at a resolution of 60,000 at m/z 400. The high collusion-induced dissociation fragmentation was performed on 28 normalized collision energy at high resolution.
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