The LC–MS analysis was performed on a Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific, San Jose, CA), coupled online to an Ultimate 3000 ultra-high performance liquid chromatography (UHPLC) instrument (Thermo Scientific, San Jose, CA). The UHPLC system was equipped with an Easy Spray Pepmap RSLC C18 column (2 µm particle, 100 Å pore size, and dimensions: 50 µm × 15 cm, Thermo Scientific, San Jose, CA). Before sample separation on the analytical column, the lyophilized sample was resuspended in 16 µL solution containing 2% (v/v) CH3CN and 0.1% (v/v) FA solution. Next, 5 µL sample was injected and loaded on an Acclaim Pepmap 100 C18 precolumn (3 µm particle size, 100 Å pore size, nanoviper, and dimensions: 75 µm × 2 cm, Thermo Scientific, San Jose, CA) at a flow rate of 5 μL/min. Sample separation was performed using a 95 min gradient. Mobile phase A consisted of 99.9% H2O and 0.1% (v/v) FA and mobile phase B of 19.92% H2O, 80% (w/v) CH3CN and 0.08% (v/v) FA. Mobile phase B increased from 4 to 10% in 5 min, 10–25% in 50 min, 25–45% in 18 min followed by a steep increase to 95% in 1 min. A flow rate of 300 nL/min was used. An inherent rinse step (10 min gradient, from 4–95% in 5 min) was applied after every 95 min separation gradient.
Acclaim pepmap100 c18 pre column
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Acclaim PepMap100 C18 pre-column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of peptides. The core function of this pre-column is to provide sample cleanup and concentration prior to the main analytical column, improving chromatographic performance and sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Acclaim pepmap rslc column, by Thermo Fisher Scientific (3 mentions)
Thermo dionex ultimate 3000, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (2 mentions)
Qtof impact 2 mass spectrometer, by Bruker (2 mentions)
Easy spray pepmap rslc c18 column, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Compass data analysis version 4, by Bruker (1 mentions)
Exploris 480, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200, by Thermo Fisher Scientific (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Compass dataanalysis, by Bruker (1 mentions)
Nanoflow hplc system, by Thermo Fisher Scientific (1 mentions)
8 protocols using acclaim pepmap100 c18 pre column
1
LC-MS Analysis of Biomolecules
2
Acetonitrile-Based LC-MS/MS Proteomics Protocol
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Acetonitrile, LC/MS grade (Fluka, #14261-1 L) and formic acid for mass spectrometry (Fluka, #94318-50ML-F) were used for preparation of buffers A and B for liquid chromatography. Buffer A: 0.1% formic acid; Buffer B: 90% acetonitrile/10% H2O/0.1% formic acid. The LC–MS/MS analysis was conducted with a nanoflow HPLC system (Thermo Dionex Ultimate 3000, Thermo Scientific) equipped with Acclaim PepMap100 C18 pre-column (5 mm × 300 µm; 5 µm particles; Thermo Scientific, #160454) and Acclaim Pep-Map RSLC column (15 cm × 75 µm, C18, 2 µm, 100 Å particles; Thermo Scientific, #164534). The Dionex UltiMate 3000 RSLCnano System was coupled to an Impact II QTOF tandem mass spectrometer (Bruker Daltonics) via a CaptiveSpray nanoBooster ion source. Acetonitrile (R Chromasolv) for liquid chromatography (Sigma–Aldrich, #34881-2.5 L) was used with the CaptiveSpray nanoBooster system. Raw data were inspected and analyzed with the Bruker Compass DataAnalysis (version 4.3) software.
3
Peptide Separation and Mass Spectrometry
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The samples, 3 µL, were analyzed on hybrid mass spectrometer Exploris 480 (Thermo Fisher Scientific) coupled with an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). A two-column setup was used on the HPLC system and peptides were loaded into an Acclaim PepMap 100 C18 precolumn (75 μm × 2 cm, Thermo Scientific) and then separated on an EASY spray column (75 μm × 50 cm, nanoViper, C18, 2 μm, 100 Å) with the flow rate of 300 nL/min. The column temperature was set to 60°C. Solvent A (0.1% FA in water) and solvent B (0.1% FA in 80% ACN) were used to create gradient, and 120-minute linear gradient from 3 to 38% of solvent B in solvent A was used to elute the peptides.
4
LC-MS/MS Analysis of Peptides
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Acetonitrile (ACN), LC/MS-grade (Carlo Erba, Val-de-Reuil Cedex, France #412342), and formic acid (FA) for mass spectrometry (Fluka, Steinheim, Germany, #94318-50ML-F) were used for the preparation of buffer A and buffer B as eluents during liquid chromatography. Buffer A: 0.1% FA; buffer B 90% ACN/10% in water, in 0.1% FA. The LC–MS/MS analysis was conducted with a nanoflow HPLC system (Thermo Dionex Ultimate 3000, ThermoScientific, Bremen, Germany) with an Acclaim PepMap100 C18 pre-column, 5 mm × 300 µm, 5 µm particles (Thermo Scientific, #160454), and an Acclaim Pep-Map RSLC column, 15 cm × 75 µm, 2 µm particles (Thermo Scientific, #164534). The MS/MS analysis utilized a QTOF Impact II mass spectrometer (Bruker, Bremen, Germany). Raw LC–MS/MS data were interpreted with the Bruker Compass Data Analysis (version 4.3) software (Bruker Daltonik GmbH, Germany).
5
Nano-LC–MS/MS Proteomic Analysis
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Samples were analyzed by nano-LC–MS/MS
on a Q Exactive Plus, coupled to an Easy-nLC 1200 (both Thermo Fisher
Scientific). The LC system was equipped with an AcclaimPepMap 100
C18 precolumn (Thermo Fisher Scientific, 3 μm particle size,
75 μm inner diameter × 2 cm length) and a nanoscale analytical
column (Thermo Fisher Scientific, AcclaimPepMap 100 C18 main column,
2 μm particle size, 75 μm inner diameter × 25 cm
length). Samples were separated using a binary gradient (solvent A:
0.1% formic acid, solvent B: 0.1% formic acid and 84% acetonitrile)
ranging from 0 to 40% in 30 min at a flow rate of 300 nL/min. MS scans
were acquired from m/z 350 to 1500
at a resolution of 70,000 with an automatic gain control (AGC) of
1e6 and a maximum injection time of 50 ms. The seven most intense
parent ions with charge states of +2 to +4 were isolated with a window
of m/z 1.2, an AGC of 5e4, and a
maximum injection time of 220 ms and fragmented with a normalized
collision energy of 28 using a dynamic exclusion of 5 s.
on a Q Exactive Plus, coupled to an Easy-nLC 1200 (both Thermo Fisher
Scientific). The LC system was equipped with an AcclaimPepMap 100
C18 precolumn (Thermo Fisher Scientific, 3 μm particle size,
75 μm inner diameter × 2 cm length) and a nanoscale analytical
column (Thermo Fisher Scientific, AcclaimPepMap 100 C18 main column,
2 μm particle size, 75 μm inner diameter × 25 cm
length). Samples were separated using a binary gradient (solvent A:
0.1% formic acid, solvent B: 0.1% formic acid and 84% acetonitrile)
ranging from 0 to 40% in 30 min at a flow rate of 300 nL/min. MS scans
were acquired from m/z 350 to 1500
at a resolution of 70,000 with an automatic gain control (AGC) of
1e6 and a maximum injection time of 50 ms. The seven most intense
parent ions with charge states of +2 to +4 were isolated with a window
of m/z 1.2, an AGC of 5e4, and a
maximum injection time of 220 ms and fragmented with a normalized
collision energy of 28 using a dynamic exclusion of 5 s.
6
Nano-LC-MS/MS Proteomics Workflow
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Tryptic peptides were separated by nano-Liquid chromatography (nLC, Dionex ultimate 3000) coupled to mass spectrometry (MS, LTQ-Orbitrap Velos; Thermo scientific) on LC analytical columns with an Acclaim PepMap 100 C18 pre-column (Thermo scientific, 100 μm x 2 cm nano Viper, 100 Å, 5 μm, P/N 164564) followed by PepMap RSLC C18 peptide separating column (Thermo scientific, 75 μm x 15 cm, 100 Å, 3 μm, P/N ES800) with 120 min gradient using buffer A (0.1% formic acid, 3%DMSO (v/v)) and 1 to 35% of buffer B (80% acetonitrile, 3% DMSO, 0.08% formic acid (v/v)). A parent ion scan was performed in the Orbitrap, using a resolving power of 60000. CID was performed with collision energy 35% and activation time of 10 ms and the top 20 most intense peptides were selected for MS/MS.
7
Quantitative LC-MS/MS Analysis of Biotinylated and Propionylated Peptides
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Cell culture, transient transfection, and sample preparation steps were described earlier [28 (link),34 (link)].
The peptide mixtures were analyzed using two LC-MS/MS systems:
The peptide mixtures were analyzed using two LC-MS/MS systems:
Nanoflow HPLC system (Thermo Dionex Ultimate 3000, ThermoScientific) with Acclaim PepMap100 C18 pre-column, 5 mm × 300 μm; 5 μm particles (Thermo Scientific, #160454) and Acclaim Pep-Map RSLC column 15 cm × 75μm, 2 μm particles (Thermo Scientific, #164534) coupled via CaptiveSpray to the QTOF Impact II mass spectrometer (Bruker). Raw LC-MS/MS data were interpreted with the Bruker Compass DataAnalysis (version 4.3) software. The separation gradient was 48 min from 2% to 50% acetonitrile. Flow rates—300 nL/min.
Nano-HPLC (Agilent Technologies 1200) was coupled to an ion-trap mass spectrometer (Bruker 6300 series) equipped with a nanoelectrospray source via protein HPLC Chip (Agilent Technologies, G4240-62001) with 40 nL trap 75 um × 43 mm 5 um 300SB-C18-ZX and analytical column packed with ZORBAX 300SB-C18, 5 µm particle size. The separation gradient was 7 min from 5% to 90% acetonitrile. Flow rates—300 nL/min.
8
Plasma Proteome Analysis by LC-MS/MS
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The plasma samples were analyzed on Hybrid mass spectrometer Q Exactive HF-X (Thermo Fischer Scientific) coupled with an Ultimate 3000 UHPLC system (Thermo Fischer Scientific). Two-column setup was used on the HPLC system, and peptides were loaded into an Acclaim PepMap 100 C18 precolumn (75 μm × 2 cm; Thermo Scientific, Waltham, MA) and then separated on an EASY spray column (75 μm × 50 cm, nanoViper, C18, 2 μm, 100 Å) with the flow rate of 300 nL·min−1. The column temperature was set to 60°C. Solvent A (0.1% FA in water) and solvent B (0.1% FA in 80% ACN) were used to create a gradient, and a 90-min linear gradient from 4% to 38% of solvent B in solvent A was used to elute the peptides.
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