The tandem mass spectrometry analysis was modified from reference56 (link). In brief, the sample re-dissolved in water was analyzed on an on-line Q Exactive mass spectrometer coupled to an EASY-nano-LC 1200 system (Thermo Fisher Scientific, MA, USA). 1.5 μL of peptide was loaded on a trap column (Thermo Fisher Scientific Acclaim PepMap C18, 100 μm × 2 cm) and an analytical column (Acclaim PepMap C18, 75 μm × 15 cm). The samples were separated with a 60 min linear gradient, from 5% B (B: 0.1% formic acid in ACN) to 35% B. The column flow rate was set as 300 nL/min at 40 °C. The electrospray voltage of 2 kV versus the inlet of the mass spectrometer was used. The mass spectrometer was run under data dependent acquisition mode, and automatically switched between MS and MS/MS mode. The full scan range was set between 300 and 1800 m/z with a resolution of 70,000, an AGC target of 3e6, a maximum injection time of 60 ms and a dynamic exclusion time of 10 s. The MS/MS scan was performed at 17,500 resolution with an AGC target of 5e4, a maximum injection time of 80 ms and a collision energy of 27.
Easy nanolc 1200 system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EASY-nanoLC 1200 system is a high-performance liquid chromatography (HPLC) instrument designed for nanoflow applications. It provides precise flow control and reliable performance for proteomic and other analytical applications requiring low sample volumes and flow rates.
Automatically generated - may contain errors
Lab products found in correlation
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (5 mentions)
Acclaim pepmap c18, by Thermo Fisher Scientific (5 mentions)
Q exactive plus, by Thermo Fisher Scientific (4 mentions)
Q exactive plus mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Peaks studio version 10, by Bioinformatics Solutions (3 mentions)
Q exactive, by Thermo Fisher Scientific (3 mentions)
Peaks studio version x, by Bioinformatics Solutions (2 mentions)
Orbitrap fusion lumos tribrid mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Orbitrap q exactive hf x mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Spectronaut 14, by Biognosys (2 mentions)
Orbitrap exploris 480 mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Easy spray acclaim pepmap c18 column, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion tribrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion lumos tribrid, by Thermo Fisher Scientific (1 mentions)
Orbitrap exploris 480, by Thermo Fisher Scientific (1 mentions)
Irt kit, by Biognosys (1 mentions)
C18 beads, by Thermo Fisher Scientific (1 mentions)
Savant spd1010, by Thermo Fisher Scientific (1 mentions)
C18 trap column, by Thermo Fisher Scientific (1 mentions)
31 protocols using easy nanolc 1200 system
1
Tandem Mass Spectrometry for Peptide Analysis
2
Quantitative Proteomics by Orbitrap Fusion
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Depleted serum fractions of each group were run in duplicates on Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific). Peptide samples (~ 1ug) were loaded on reverse phase EASY-Spray Acclaim PepMap C18 Column (15 cm × 75 μm I.D., packed with C18 resin, three μm particle size, 100 Å; Thermo Scientific, Part No ES800), coupled to Easy NanoLC 1200 system (Thermo Fisher Scientific, San Jose, US). The peptide separation was achieved with a constant flow rate of 300 nl/min using a linear gradient of 5–95% solvent B (80% ACN in 0.1% formic acid) with solvent A (2% ACN in 0.1% formic acid) for 95–100 min. MS master scan was acquired at Orbitrap mass analyzer in the m/z range of 375–1600 with a mass resolution of 120,000 at m/z 200. Data-dependent MS/MS acquisition was achieved by IonTrap mass analyzer with 3s as cycle time. Quadrupole with an isolation window of 1.2 m/z was selected as an isolation mode. AGC target was set to 5000, and the maximum injection time was 45 ms. The precursor ion fragmentation with charge states 2–5 was achieved using higher-energy collisional dissociation at 30% collision energy. The lock mass option was enabled for real-time calibration using polycyclodimethylsiloxane (m/z, 445.12) ions.
3
Proteomic Profiling of APAP-Induced Liver Injury
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WT and GSDME‐KO mice were sacrificed 12 h after injection of APAP. Liver tissues were lysed and immunoprecipitated with an antibody against ISG15. The immunoprecipitated samples were separated by SDS‐PAGE to distinguish the differential band between WT‐APAP and GSDME‐KO‐APAP. The gel containing the band of interest was excised and subjected to in‐gel digestion with trypsin. The resulting peptides were analyzed by Nano‐HPnano‐LC‐MS/MS on an EASY‐nanoLC 1200 system coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA). Tandem mass spectra were processed using PEAKS Studio version X+ (Bioinformatics Solutions Inc., Waterloo, Canada). PEAKS DB was set up to search the UniProt_Mus musculus database (version 201 907, with 22 290 entries) assuming trypsin as the digestion enzyme. Peptides with a −10lgP score greater than or equal to 20 and proteins with a −10lgP score greater than or equal to 20, and containing at least one unique peptide, were filtered for further analysis.
4
Quantitative Proteomics via Orbitrap Fusion
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The protein samples were passed through an Orbitrap Fusion™Lumos™Tribrid™ mass spectrometer (Thermo Fisher Scientific) with an Easy-NanOLc 1200 system (Thermo Fisher Scientific) online LC–MS/MS analysis of nanosprays. The samples were loaded with 2 μL peptide (analytical column: Acclaim PepMap C18, 75 μm × 25 cm) and separated by gradient for 60 min. The column temperature was 40 °C and the flow rate was maintained at 300 nL/min. Use an electrospray voltage of 2 kv on the inlet of the mass spectrometer.
5
Proteomic Analysis by DDA and DIA Mass Spectrometry
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DDA and DIA analyses were performed using a Q-Exactive mass spectrometer (Thermo) coupled with an Easy-nanoLC 1200 system. The peptide digests were reconstituted in nano-LC mobile phase A (0.1% formic acid), loaded onto a NanoViper C18 trap column (3 mm, 100 Å), and separated on an analytical column (75 mm × 25 cm C18-2 mm 100 Å) via a 2-hr linear gradient from 5 to 35% mobile phase B (95% ACN with 0.1% formic acid) at a flow rate of 300 nL/min, followed by a linear increase to 95% mobile phase B for 2 min and 95% for 6 min. The Q-Exactive mass spectrometer was operated with a spray voltage of 1.9 kV and a capillary temperature of 275°C in a 350–1500 m/z scan range and 70,000 resolution, and the maximum ion injection time was 100 ms. Twenty of the most abundant precursor ions from each DDA cycle were selected for higher-energy collision dissociation fragment analysis with a maximum ion injection time of 50 ms, collision energy of 28 eV, and dynamic exclusion of 25 s. For each sample in the DIA analysis, MS1 and MS2 were set with a maximum ion injection time of 50 ms. MS1 was set as mentioned above (350–1500 m/z scan range and 70,000 resolution) and was separated into 34 acquisition windows with a mass range of 14–152 Da. The liquid conditions were consistent with the DDA model for separation. Raw DIA data were analyzed using Spectronaut Pulsar X.
6
Peptide Identification by DIA-MS
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The peptides were redissolved in solvent A (A: 0.1% formic acid in water) and analyzed by an Orbitrap Fusion Lumos Tribrid coupled to an EASY-nanoLC 1200 system (Thermo Fisher Scientific, MA, USA). Next, 4 μL of the peptide sample was loaded onto a 25 cm analytical column (75 μm inner diameter, 1.9 μm resin (Dr Maisch)) and separated by gradient applied over 90 min as follows: 4% buffer B (80% ACN with 0.1% FA) for 3 min; a stepwise increase to 50% buffer B in 82 min; increase to 95% buffer B in 1 min; and a hold for 7 min. The column flow rate was maintained at 250 nL/min, and the column temperature was 55°C. The electrospray voltage was set to 2 kV. The mass spectrometer was run under data independent acquisition (DIA) mode with hybrid data strategy. A survey scan was acquired at 120,000 resolution, normalized AGC target of 250% and a maximum injection time of 100ms. In the DIA MS2 acquisition, variable Isolation window were performed. One full scan followed by 20 windows with resolution of 50,000, normalized AGC target of 200%, a maximum injection time of 86ms and normalized collision energy at 33.
7
Peptide Analysis by LC-MS
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The peptides were redissolved in solvent A (A: 0.1% formic acid in water) and analysed by LC-MS which consists of EASY-nano LC 1200 system (Thermo Fisher Scientific, USA) and Orbitrap Exploris 480 (Thermo Fisher Scientific, USA). 3 µL sample was loaded into analytical column (Acclaim PepMap C18, 75 μm × 25 cm, Dionex, USA) and separated with 130 min-gradient. A flow rate of 250 nL/min was maintained using a linear ACN gradient of 2~8% solvent B in 6 min followed by 8% to 35% solvent B in the next 124 min (solvent A: 99.9% H2O, 0.1% formic acid; solvent B: 99.9% ACN, 0.1% formic acid). The mass spectrometer was operated under the data-dependent acquisition mode. The parameters were set as follows: (i) The electrospray voltage was set 2 kV; (ii) MS: scan range (m/z) = 350-1200; resolution=120,000; AGC target=300; maximum injection time=50 ms; (iii) HCD-MS/MS: resolution=30,000; AGC target=200; collision energy=35.
8
Amino Acid Sequence and Mass Analysis
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The amino acid sequence and the molecular mass of H1‐2were analyzed by (Qingdao Sci‐tech innovation Co., Ltd.) with online nanospray LC‐MS/MS on a Q Exactive Plus coupled to an EASY‐nano‐LC 1200 system (Thermo Fisher Scientific). Five microliters of the sample was loaded into the trap column (Thermo Fisher Scientific Acclaim PepMap C18, 100 μm × 2 cm), with a flow rate of 10 μL/min, and subsequently separated on the analytical column (Acclaim PepMap C18, 75 μm × 15 cm), with a linear gradient, from 3% to 38% B (A: distilled water, B: 0.1% formic acid in ACN) in 60 min. The column flow rate was maintained at 0.5 ml/min with the column temperature of 40°C. The peptide sequences of the major peaks were performed by processing the ion series in MS/MS spectra using Peaks Studio 8.5 and manual interpretation.
A spray voltage of 2 kV was applied, and the instrument, Q Exactive Plus, was operated under switching automatically between MS full scan and data‐dependent fragmentation modes with 27 of collision energy. Ions were scanned at high resolution (70,000 in MS1, 17,500 in MS2), and the MS scan range was 100–1500 m/z at both MS1 and MS2 levels.
A spray voltage of 2 kV was applied, and the instrument, Q Exactive Plus, was operated under switching automatically between MS full scan and data‐dependent fragmentation modes with 27 of collision energy. Ions were scanned at high resolution (70,000 in MS1, 17,500 in MS2), and the MS scan range was 100–1500 m/z at both MS1 and MS2 levels.
9
Nano-LC-MS Peptide Separation Protocol
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Vacuum-dried tryptic peptide pellets were re-dissolved in 10 to 20 μL of 0.1% Formic acid (FA), vortexed gently and prior to injection centrifuged at 12000 RPM for 15 min. Around 3–5 μL of the sample was bound onto a pre-equilibrated Acclaim PepMap 100, 75 um x 2 cm, Nanoviper, C18 pre-column (Thermo Fisher Scientific, USA) at a flow rate of 4 μL/min. Reverse phase separation of the peptide mixture was performed using the Easy Nano-LC 1200 system (Thermo Fisher Scientific, USA) using a PepMap RSLC C18—Easy spray Analytical column of 75um x 50cm length at a flow rate of 250 to 300 nL/min with a solvent system comprising solvent A (0.1% FA), and solvent B (0.1%FA in 80% ACN, v/v). The column temperature was maintained at 40°C throughout the run. The gradient conditions were set to achieve 5 to 8% B for 2 min; followed by a linear increase of 8% to 20% B for 150 min, 20% to 40% B for 10 min, 40% to 80% B for 5 min, wash with 80% B for 5mins, 80% to 5% B for 2 min and 5% B for 6 min.
10
Nano-LC-MS/MS Profiling of Peptide Mixtures
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Two groups of Peptide mixtures (sample and control, three replicates) were tested by Easy nano-LC 1200 system (Thermo Scientific, San Jose, CA, USA) using a reverse-phase column (ReproSil-Pur C18-AQ (75 μm × 3 cm, 2 μm)) with a 120-min gradient at a flow rate of 200 nL/min. MS profiling spectra were obtained on a Q-Exactive Plus mass spectrometer (Thermo Scientific, San Jose, CA, USA). The mobile phases A and B were 0.1% formic acid in LC/MS-grade water and 0.1% formic acid in 80% acetonitrile, respectively. The peptides were separated with a linear gradient 3–32% B in 95 min, 32%–100% B in 10 min, and 100% B in 15 min. Full scans were acquired over a mass range of 355–1700 at the resolution of 70,000. The MS/MS scans were acquired at a resolution of 35,000.
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