Dried fractions were reconstituted in 3% MeCN/0.1% FA to an estimated peptide concentration of 1 µg/µl and analyzed via coupled nanoflow liquid chromatography and tandem mass spectrometry (LC-MS/MS) using a Proxeon Easy-nLC 1200 (Thermo Fisher Scientific) coupled to an Orbitrap Exploris 480 Mass Spectrometer (Thermo Fisher Scientific). A sample load of 1 µg for each fraction was separated on a capillary column (360x75 µm, 50 °C) containing an integrated emitter tip packed to a length of approximately 25 cm with ReproSil-Pur C18-AQ 1.9 μm beads (Dr. Maisch GmbH). Chromatography was performed with a 110 min gradient of solvent A (3% MeCN/0.1% FA) and solvent B (90% MeCN/0.1% FA). The gradient profile, described as min:% solvent B, was 0:2, 1:6, 85:30, 94:60, 95:90, 100:90, 101:50, 110:50. Ion acquisition was performed in data-dependent mode with the following relevant parameters: MS1 orbitrap acquisition (60,000 resolution, 350–1800 scan range (m/z), 300% normalized AGC target, 25ms max injection time) and MS2 orbitrap acquisition (20 scans per cycle, 0.7 m/z isolation window, 32% HCD collision energy, 45,000 resolution, 50% normalized AGC target, 50ms max injection time, 15 s dynamic exclusion, 50% fit threshold, and 1.2 m/z fit window).
Reprosil pur c18 aq 1.9 μm beads
Manufactured by Dr. Maisch
ReproSil-Pur C18-AQ 1.9 μm beads are a type of chromatographic packing material used in high-performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC) applications. The beads are made of silica and have a C18 alkyl bonded phase, which provides a stationary phase for the separation and analysis of a wide range of organic compounds. The bead size is 1.9 micrometers, which allows for high-resolution separations and increased efficiency in HPLC and UHPLC systems.
Automatically generated - may contain errors
Lab products found in correlation
Easy nlc 1200, by Thermo Fisher Scientific (4 mentions)
Proxeon easy nlc 1200, by Thermo Fisher Scientific (2 mentions)
Orbitrap exploris 480 mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Q exactive hf x hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (1 mentions)
23 cm fused silica emitter, by New Objective (1 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (1 mentions)
Q exactive plus hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
7 protocols using reprosil pur c18 aq 1.9 μm beads
1
Tandem Mass Spectrometry Proteome Profiling
2
Proteomic Analysis by Q-Exactive HF MS
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For each sample, 1 μg of peptides was injected in a Q-exactive HF mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a linear gradient from 95% solvent A (2% acetonitrile and 0.1% formic acid) to 55% solvent B (80% acetonitrile and 0.1% formic acid) over 120 min, followed by 100% of solvent B in 3 min at a constant flow rate of 0.25 μl/min on ultra-high-performance liquid chromatograhphy (UHPLC) Easy-nLC 1000 (Thermo Fisher Scientific). The LC system was connected to a 23-cm fused silica emitter of 75-μm inner diameter (New Objective, Inc), packed in-house with ReproSil-Pur C18-AQ 1.9-μm beads (Dr Maisch Gmbh) using a high-pressure bomb loader (Proxeon). A data-dependent acquisition was performed with the following settings: enabled dynamic exclusion of 20 s, MS1 resolution of 60,000 at m/z 200, MS1 automatic gain control target of 3e+6, MS1 maximum fill time of 20 ms, MS2 resolution of 15,000, MS2 automatic gain control target of 1e+5, MS2 maximum fill time of 80 ms, and MS2 normalized collision energy of 28. For each data-dependent acquisition cycle, one full MS1 scan range of 300–1,650 m/z was followed by 12 MS2 scans, using an isolation window size of 2 m/z. The resulting proteomic data have been loaded into PeptideAtlas repository (Dataset Identifier PASS01302 ).
3
High-Resolution Mass Spectrometry Proteomics
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All the samples were analyzed on a Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer coupled with an Easy nLC 1200 ultra-high pressure liquid chromatography system (Thermo Fisher Scientific) with solvent A of 0.1% formic acid and solvent B of 0.1% formic acid/80% acetonitrile. 1 μg of each sample was injected on a 75-μm ID PicoFrit column packed in-house to approximately 30-cm length with ReproSil-Pur C18-AQ 1.9-μm beads (Dr. Maisch). Column equilibration and peptide loading were done at 900 bars in buffer A (0.1% FA). Samples were separated at a 250 nL/min flow rate with a gradient of 2–7% buffer B in 5min, 23% buffer B in 70min, 23 to 45% buffer B in 30min, 45 to 95% buffer B in 5min, followed by a hold at 95% for 7min and back to 2% for 15min. Column temperature was set to 60°C. Mass spectrometer was operated in data-dependent acquisition mode. The MS instrument parameters were set as follows: MS1, r = 70,000; MS2, r = 17,500; MS1 AGC target of 3e6; MS2 for the 10 most abundant ions using an AGC target of 1e6 and maximum injection time of 60ms; and a 45s dynamic exclusion. The isolation window was set to 1.6m/z and normalized collision energy fixed to 28 for HCD fragmentation. Unassigned precursor ion charge states as well as 1, 7, 8 and >8 charged states were rejected and peptide match was disable.
4
K-GG Peptide LC-MS/MS Analysis
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Reconstituted K-GG enriched peptides were analyzed via coupled nanoflow liquid chromatography and tandem mass spectrometry (LC-MS/MS) using a Proxeon Easy-nLC 1200 (Thermo Fisher Scientific) coupled to an Orbitrap Exploris 480 Mass Spectrometer (Thermo Fisher Scientific) equipped with a FAIMS interphase. Four out of 9 μl of total eluted material was separated on a capillary column (360x75 µm, 50 °C) containing an integrated emitter tip packed to a length of approximately 25 cm with ReproSil-Pur C18-AQ 1.9 μm beads (Dr. Maisch GmbH). Chromatography was performed with a 154 min gradient of solvent A (3% MeCN/0.1% FA) and solvent B (90% MeCN/0.1% FA). The gradient profile, described as min:% solvent B, was 0:2, 2:6, 122:35, 130:60, 133:90, 143:90, 144:50, 154:50. Ion acquisition was performed in data-dependent mode with the following relevant parameters: three FAIMS CV settings (–45 V, –50 V, and –70 V), MS1 orbitrap acquisition (60,000 resolution, 350–1800 scan range (m/z), 100% normalized AGC target, 10ms max injection time) and MS2 orbitrap acquisition (10 scans per cycle, 0.7 m/z isolation window, 32% HCD collision energy, 45,000 resolution, 50% normalized AGC target, 120ms max injection time, 20 s dynamic exclusion, 50% fit threshold, and 1.4 m/z fit window).
5
Orbitrap-based Shotgun Proteomics Protocol
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Desalted peptides were resuspended in 9 μl of 3% ACN/0.1% FA and analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) using a Proxeon Easy-nLC 1200 coupled to a Q Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). A 4-μl of sample from each fraction was separated on a capillary column (360 × 75 μm, 50°C) containing an integrated emitter tip packed to a length of approximately 25 cm with ReproSil-Pur C18-AQ 1.9-μm beads (Dr. Maisch GmbH). Chromatography was performed with a 110-min gradient of solvent A (3% ACN/0.1% FA) and solvent B (90% ACN/0.1% FA). The gradient profile, described as min:% solvent B, was 0:2, 1:6, 85:30, 94:60, 95:90, 100:90, 101:50, and 110:50. Ion acquisition was performed in data-dependent MS2 (ddMS2) mode with the following relevant parameters: MS1 acquisition (60,000 resolution, 3E6 AGC [automatic gain control] target, 10 ms max injection time) and MS2 acquisition (loop count = 20, 0.7 m/z isolation window, 31 NCE [normalized collision energy], 45,000 resolution, 5E4 AGC target, 105 ms max injection time, 1E4 intensity threshold, 15-s dynamic exclusion, and charge exclusion for unassigned, 1 and >6).
6
Orbitrap Exploris 480 UPLC-MS/MS Protocol
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Samples were analyzed on an Orbitrap Exploris 480 mass spectrometer coupled with Easy-nLC 1200 ultra-high pressure liquid chromatography (UPLC) system (Thermo Fisher Scientific) with solvent A of 0.1% formic acid (FA)/3% acetonitrile and solvent B of 0.1% FA/90% acetonitrile. Half of each of the RAP-MS fraction were injected on a 75 μm ID Picofrit column packed in-house to approximately 28 cm length with Reprosil-Pur C18-AQ 1.9 μm beads (Dr. Maisch GmbH). Samples were separated at 200 nL/min flow rate with a gradient of 2-6% solvent B for 1 min, 6-30% B in 84 min, 30-60% B in 9 min, 60-90% B in 1 min, followed by a hold at 90% B for 5 min. The mass spectrometer was operated in data-dependent acquisition mode. On Exploris 480 MS1 scan (r = 60,000) was followed by MS2 scans (r = 45,000) for top 20 most abundant ions using normalized automatic gain control (AGC) of 100% for MS1 and 200% for MS2, MS2 maximum inject time of 150 ms, normalized collision energy of 34 and fit filter of 50%.
7
Nano-LC-MS/MS Peptide Analysis
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Desalted peptides were resuspended in 9 μL of 3% ACN/0.1% FA and analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) using a Proxeon Easy-nLC 1200 coupled to a Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Four μL of sample from each fraction was separated on a capillary column (360 x 75 µm, 50 °C) containing an integrated emitter tip packed to a length of approximately 25 cm with ReproSil-Pur C18-AQ 1.9 μm beads (Dr. Maisch GmbH). Chromatography was performed with a 110 min gradient of solvent A (3% ACN/0.1% FA) and solvent B (90% ACN/0.1% FA). The gradient profile, described as min:% solvent B, was 0:2, 1:6, 85:30, 94:60, 95:90, 100:90, 101:50, 110:50. Ion acquisition was performed in data-dependent MS2 (ddMS2) mode with the following relevant parameters: MS1 acquisition (60,000 resolution, 3E6 AGC target, 10ms max injection time) and MS2 acquisition (Loop count = 20, 0.7m/z isolation window, 31 NCE, 45,000 resolution, 5E4 AGC target, 105 ms max injection time, 1E4 intensity threshold, 15s dynamic exclusion, and charge exclusion for unassigned, 1 and >6).
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