Concentrated peptides (250 ng) were separated on a nanoHPLC system (nanoElute, Bruker Daltonics, Billerica, MA, USA). The samples were loaded onto an Acclaim PepMap100 C18 Trap Column (0.3 mm id × 5 mm, Dionex Corporation, Sunnyvale, CA, USA) at 4 µL/min consistent flow and peptides were eluted onto a PepMap C18 analytical nanocolumn (1.9 µm beads size, 75 µm × 25 cm, PepSep, Marslev, Denmark) heated at 50 °C. Peptides were eluted with solvent B (100% ACN and 0.1% FA) in a 5–37% linear gradient with a flowrate of 400 nL/min for ~2 h. The HPLC system was coupled to a TimsTOF Pro ion mobility mass spectrometer containing Captive Spray nano electrospray source (Bruker Daltonics). Data acquisition was undertaken using diaPASEF mode. For each individual Trapped Ion Mobility Spectrometry (TIMS) measurement in diaPASEF mode, a single mobility window consisting of 27 mass steps (with m/z ranging from 114 to 1414 and a mass width of 50 Da) was employed per cycle, with a 1.27 s duty cycle. This process involves scanning the diagonal line in the m/z-ion mobility plane for +2 and +3 charged peptides.
Acclaim pepmap 100 c18 trap column
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Acclaim PepMap 100 C18 trap column is a reversed-phase liquid chromatography column designed for sample concentration and desalting prior to analytical separation. It features a spherical, porous silica-based stationary phase with a particle size of 5 μm and a pore size of 100 Å.
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Lab products found in correlation
Easy nlc 1200, by Thermo Fisher Scientific (6 mentions)
Acclaim pepmap 100 c18 analytical column, by Thermo Fisher Scientific (6 mentions)
Reprosil pur c18, by Dr. Maisch (5 mentions)
Acclaim pepmap rslc c18 analytical column, by Thermo Fisher Scientific (4 mentions)
Acquity uplc m class peptide beh c18 column, by Waters Corporation (4 mentions)
Orbitrap eclipse tribrid mass spectrometer, by Thermo Fisher Scientific (4 mentions)
Ultimate 3000, by Thermo Fisher Scientific (3 mentions)
Dionex ultimate 3000 system, by Thermo Fisher Scientific (3 mentions)
Halo fused core particles, by Merck Group (2 mentions)
Ultimate 3000 nanouplc, by Thermo Fisher Scientific (2 mentions)
Dionex ultimate 3000 nanorslc, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 rslcnano, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion tribrid mass, by Thermo Fisher Scientific (2 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 nano rslc system, by Thermo Fisher Scientific (2 mentions)
Maxis 2 etd q tof, by Bruker (2 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Easy nlc 1200 uplc system, by Thermo Fisher Scientific (2 mentions)
Q exactive hf, by Thermo Fisher Scientific (2 mentions)
35 protocols using acclaim pepmap 100 c18 trap column
1
Peptide Profiling by nanoHPLC-TIMS-MS
2
Proteomic Analysis of IgG and PR3-ANCA by nanoLC-MS
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The digested samples were analyzed by nanoLC-reversed phase (RP)-electrospray ionization (ESI) – quadrupole time-of-flight (qTOF)-MS on an Ultimate 3000 HPLC system (Dionex/Thermo Scientific, Sunnyvale, CA) coupled to a MaXis Impact (Bruker Daltonics, Bremen, Germany). The samples were concentrated on a Dionex Acclaim PepMap100 C18 trap column (particle size 5 μm, internal diameter 300 μm, length 5 mm) and separated on an Ascentis Express C18 nano column (2.7 μm HALO fused core particles, internal diameter 75 μm, length 50 mm; Supelco, Bellefonte, PA). The following linear gradient was applied, with solvent A consisting of 0.1% trifluoroacetic acid (TFA; Fluka) and solvent B of 95% acetonitrile (Biosolve, Valkenswaard, The Netherlands): t = 0 min, 3% solvent B; t = 2, 6%; t = 4.5, 18%; t = 5, 30%; t = 7, 30%; t = 8, 1%; t = 10.9, 1%. The sample was ionized in positive ion mode with a CaptiveSprayer (Bruker Daltonics) at 1100 V. A nanoBooster (Bruker Daltonics) was used to enrich the nitrogen gas with acetonitrile to enhance the ionization efficacy. A mass spectrum was acquired every second (frequency of 1 Hz), with the ion detection window set at m/z 550–1800. In between every 12 measurements an external IgG standard was run. A mass spectrum of both total IgG and PR3-ANCA can be seen in Fig. 1 .
3
Nano-LC-MS/MS Peptide Analysis
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The sample was loaded to the column equilibrated with buffers A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). The Acclaim PepMap 100 C18 trap column (75 μm × 2 cm, Dionex) was equilibrated with liquid A by Ultimate 3000 nanoUPLC (Dionex), and the sample was eluted onto an Acclaim PepMap RSLC C18 analytical column (75 μm × 25 cm, Dionex) at a flow rate of 300 nl/min. The liquid-phase gradient was given as follows: 0–6 min, where the linear gradient of buffer B was from 2 to 10%; 7–51 min, where the linear gradient of buffer B was from 10 to 20%; 51–53 min, where the linear gradient of buffer B was from 20 to 80%; 53–57 min, where the linear gradient of buffer B solution was held at 80%.
Mass analysis was performed by nano-spray ionization-mass spectrometry (NSI-MS) and Q-Exactive HF mass spectrometry (Thermo Scientific). The intact peptide was detected by the Orbitrap, the scanning range was set to 250–1,500 m/z, the automatic gain control (AGC) target was 3E6, the resolution was 70,000, the max injection time (IT) was 250 ms, and the dynamic exclusion time was 15 s. The peptide was selected and fragmented for MS/MS using 28% NCE; ion fragments were detected in the Orbitrap, the resolution was 17,500, AGC was 1E5 or 5E4, and the maximum IT was 100 or 200 ms. LC-MS was performed by Micrometer Biotech (Hangzhou, China).
Mass analysis was performed by nano-spray ionization-mass spectrometry (NSI-MS) and Q-Exactive HF mass spectrometry (Thermo Scientific). The intact peptide was detected by the Orbitrap, the scanning range was set to 250–1,500 m/z, the automatic gain control (AGC) target was 3E6, the resolution was 70,000, the max injection time (IT) was 250 ms, and the dynamic exclusion time was 15 s. The peptide was selected and fragmented for MS/MS using 28% NCE; ion fragments were detected in the Orbitrap, the resolution was 17,500, AGC was 1E5 or 5E4, and the maximum IT was 100 or 200 ms. LC-MS was performed by Micrometer Biotech (Hangzhou, China).
4
Peptide Characterization by Nanoflow LC-MS/MS
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The lyophilized peptide was resuspended in buffer A (2% ACN, 0.1% FA), loaded onto an Acclaim PepMap 100 C18 trap column (75 μm × 2 cm, Dionex, Waltham, MA, USA) with Ultimate 3000 nanoUPLC (Dionex) and eluted onto an Acclaim PepMap RSLC C18 analytical column (Dionex, 75 μm × 25 cm). Buffer A linear gradient was run at 300 nL min−1 for 45 min, starting from 11% to 20% buffer B, followed by 2 min gradient to 80% buffer B, and maintained at 80% buffer B for 3 min. The peptide was subjected to NSI source in mass spectrometry Q Exactive plus (Waltham, MA, USA) coupled online to the UPLC. MS1 spectra were acquired at the resolution of 70 000 (at 200 m/z) with an AGC (automatic gain control) of 3 000 000 and a max IT of 250 ms. MS/MS data were acquired at the resolution of 17 500 through isolation windows of 2 Da, and the AGC and max IT were set to 100 000 and 100 ms, the NCE (normalized collision energy) was set to 28%. And the loop count was set to 15 which means 15 MS/MS scans would be acquired between each full scan.
5
Quantitative Peptide Analysis by HPLC-MS/MS
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The labeled peptide was resuspended in buffer A (2% ACN, 0.1% FA) and centrifuged at 20,000×g for 2 min. The supernatant was transferred into a sample tube and loaded onto an Acclaim PepMap 100 C18 trap column (75 µm×2 cm; Dionex) by EASY nLC1000 nano UPLC (Thermo). The peptide was then eluted onto an Acclaim PepMap RSLC C18 analytical column (50 µm×15 cm; Dionex). An 85 min gradient program was run at 300 nL/min, which started from 3% to 35% B (80% ACN, 0.1% FA), followed by 5 min linear gradient to 90% B, and maintained at 90% B for 5 min.
The peptides were subjected to NSI source, followed by tandem mass spectrometry (MS/MS) in Q Exactive (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using 27% NCE with 12% stepped NCE; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied to the top 20 precursor ions above a threshold ion count of 3E4 in the MS survey scan with 5.0 s dynamic exclusion. The applied electrospray voltage was 1.8 kV. Automatic gain control was used to prevent overfilling of the ion trap; 1E5 ions were accumulated for generation of MS/MS spectra. The m/z scan range was 350 Da to 2000 Da for MS scans.
The peptides were subjected to NSI source, followed by tandem mass spectrometry (MS/MS) in Q Exactive (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using 27% NCE with 12% stepped NCE; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied to the top 20 precursor ions above a threshold ion count of 3E4 in the MS survey scan with 5.0 s dynamic exclusion. The applied electrospray voltage was 1.8 kV. Automatic gain control was used to prevent overfilling of the ion trap; 1E5 ions were accumulated for generation of MS/MS spectra. The m/z scan range was 350 Da to 2000 Da for MS scans.
6
IgG Glycopeptide Analysis by LC-MS
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The IgG glycopeptide samples were analysed using liquid chromatography coupled to mass spectrometry (LC-MS), in a setup described previously41 (link). An amount of 2.5 µL of the samples was injected in an Ultimate 3000 RSLCnano liquid chromatography system (Dionex, Sunnyvale, CA). The samples were first washed on an Acclaim PepMap100 C18 trap column (5 mm × 300 µm i.d., Dionex, Sunnyvale, CA), and subsequently separated on an Ascentis Express C18 nanoLC column (50 mm × 75 µm i.d., 2.7 µm HALO fused core particles; Supelco, Bellefonte, PA) with a flow rate of 0.9 µL/min. The following linear gradient was used, with solvent A consisting of 0.1% trifluoroacetic acid and B of 95% acetonitrile (ACN): t = 0, 3% solvent B; t = 2, 6%; t = 4.5, 18%; t = 5, 30%; t = 7, 30%; t = 8, 0%; t = 11, 0%.
Via a sheath-flow electrospray (ESI) interface (Agilent Technologies, Santa Clara, CA), the LC was coupled to a Maxis Impact quadrupole time-of-flight (QTOF)-MS system (micrOTOF-Q; Bruker Daltonics, Bremen, Germany). A sheath-flow consisting of 50% isopropanol, 20% propionic acid (Merck) and 30% MilliQ-purified water was applied at 2 µL/min, and nitrogen gas was applied at 4 L/min. MS1 spectra were acquired with a frequency of 0.5 Hz and within an m/z range of 600-2000. An IgG standard and two blank injections were run in between every 12 runs.
Via a sheath-flow electrospray (ESI) interface (Agilent Technologies, Santa Clara, CA), the LC was coupled to a Maxis Impact quadrupole time-of-flight (QTOF)-MS system (micrOTOF-Q; Bruker Daltonics, Bremen, Germany). A sheath-flow consisting of 50% isopropanol, 20% propionic acid (Merck) and 30% MilliQ-purified water was applied at 2 µL/min, and nitrogen gas was applied at 4 L/min. MS1 spectra were acquired with a frequency of 0.5 Hz and within an m/z range of 600-2000. An IgG standard and two blank injections were run in between every 12 runs.
7
Shotgun Proteomics Analysis Pipeline
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Shotgun proteomics analysis was carried out on 5 μg of tryptic digest using a Dionex Ultimate 3000 nanoRSLC (Dionex, Sunnyvale, CA, USA) coupled to a Bruker Maxis II mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) via CaptiveSpray nanobooster ionsource. Tryptic digest samples were desalted on an Acclaim PepMap100 C-18 trap column (100 μm × 20 mm, Thermo Scientific, Sunnyvale, CA, USA) using 0.1% TFA for 8 min at a flow rate of 5 μL/min and separated on the ACQUITY UPLC M-Class Peptide BEH C18 column (130 Å, 1.7 μm, 75 μm × 250 mm, Waters, Milford, MA, USA) at 300 nL/min flow rate, 48 °C column temperature. Solvent A was 0.1% formic acid, solvent B was acetonitrile, 0.1% formic acid and a linear gradient from 4% B to 50% B in 90 min was used. Mass spectrometer was operated in the data dependent mode using a fix cycle time of 2.5 s. MS spectra was acquired at 3 Hz, while MS/MS spectra at 4 or 16 Hz depending on the intensity of the precursor ion. Singly charged species were excluded from the anaylsis.
8
Proteomic Analysis by LC/MS/MS
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For proteomic analysis, we followed a previously described method for sample treatment with trypsin followed by desalting on a C18 solid-phase extraction cartridge (Wiśniewski et al., 2009 (link)). The dried sample was dissolved in 10 µl of 0.1% formic acid for LC/MS/MS analysis with the use of the Thermo Fisher Scientific LC-nESI-Q Exactive mass spectrometer coupled with online nanoUHPLC (Dionex UltiMate 3000 Binary RSLCnano). An Acclaim PepMap 100 C18 trap column (75 µm × 2.0 cm, 3 µm, 100 Å; Thermo Fisher Scientific) and an Acclaim PepMap RSLC C18 nano LC column (75 µm × 25 cm, 2 µm, 100 Å) were used to deliver solvent and separate tryptic peptides with a linear gradient from 5% to 35% acetonitrile in 0.1% (vol/vol) formic acid for 90 min at a flow rate of 300 nl/min. The acquisition cycle of the MS data were performed with the data-dependent mode with a full-survey MS scan followed by 10 MS/MS scans of the top 10 precursor ions from the MS scan. The MS scan was performed with a resolving power of 70,000 over the mass-to-charge (m/z) range of 350 to 1,600 and dynamic exclusion enabled. The data-dependent MS/MS acquisitions were performed with a 2-m/z isolation window, 27% normalized collision energy, and 17,500 resolving power. Finally, the MS data were analyzed using Proteome Discoverer 2.1 SP1 (Thermo Fisher Scientific).
9
Orbitrap LC-MS/MS Peptide Analysis
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Typically, 10 µL of sample was injected into the LC–MS/MS system consisting of a Dionex UltiMate 3000 Rapid Separation LC (RSLC) system with an Orbitrap Fusion or Q Exactive Plus Orbitrap (Thermo Scientific). Peptides were trapped using an Acclaim PepMap100 C18 Trap column (Thermo Scientific) at a flow rate of 15 μL/min using 0.1% formic acid/2% acetonitrile in H2O (solvent A). Peptides were eluted from the trap column to an in-house packed column (75 µm inner diameter (ID) × 25 cm; 1.9 µm C18 media, Dr Maisch) with a pulled tip emitter. Peptides were eluted from the column using a linear 100-min gradient from 5% to 40% solvent B (0.1% formic acid/80% acetonitrile). Data was collected in positive mode and full spectra acquired from m/z 350 to 2000, with a resolution of 70,000. For MS/MS, the top 15 most intense precursor ions were fragmented and ionised, and MS/MS spectra with m/z 350–1750 and resolution 17,500 recorded.
10
Orbitrap Fusion Lumos LC-MS Peptide Analysis
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LC-MS experiments were performed on an Orbitrap Fusion Lumos mass spectrometer interfaced with an Easy-nLC1200 nanoflow liquid chromatography system (both from Thermo Fisher Scientific). A total of 8 µl out of 15 μl of each peptide sample were trapped on an Acclaim Pepmap 100 C18 trap column (100 μm × 2 cm, particle size 5 μm, Thermo Fischer Scientific) and separated on an analytical column (75 μm × 35 cm) packed in-house with Reprosil-Pur C18 material (particle size 3 μm, Dr. Maisch, Germany) using a gradient with 0.2% formic acid in water as solvent A and 80% acetonitrile with 0.2% formic acid as solvent B at a flow rate of 300 nL/min. The elution profile was as follows: 5% to 33% B in 77 min, 33% to 100% B in 3 min, and 100% B for 10 min. Precursor ion scans were performed at 120,000 target resolution with an m/z range of 375–1500 and an AGC target of 4e5. The most abundant precursors with charges 2–7 were selected for fragmentation with a maximum duty cycle of 3 s and a dynamic exclusion duration of 45 s. Precursors were isolated with a 1.0 Da window and fragmented by higher energy collision-induced dissociation at 30% collision energy with a maximum injection time of 150 ms and an AGC target 5e4, and the MS2 spectra were recorded at 30,000 resolution.
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