LC-MS/MS system consisted of an Orbitrap Eclipse Tribrid Mass Spectrometer (Thermo Scientific, Waltham, MA) and a Dionex Ultimate3000 nano-UPLC system (Thermo Scientific, Waltham, MA). An Acclaim PepMap 100 (C18, 5 μm, 100 Å, 100 μm x 2 cm) trap column and an Acclaim PepMap RSLC (C18, 2 μm, 100 Å, 75 μm x 50 cm) analytical column was be employed for peptide separation. MS spectra was acquired by parallel reaction monitoring consisting of MS/MS scans of the targeted precursor ions from the MS1 scan with dynamic exclusion option with 30 s of duration. Skyline (MacCoss Lab Software, University of Washington, Seattle, WA) was used for target peptides detection, peak feature extraction, and peak area calculation for quantitative data analysis. Peak areas were normalized using the total ion current (TIC) which is the sum of all peaks in the chromatogram acquired by complementary MS1 scan event. NIST mass spectral library was utilized to confirm peak selection for the analysis.
Orbitrap eclipse tribrid mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Orbitrap Eclipse Tribrid mass spectrometer is a high-resolution, accurate-mass (HRAM) instrument designed for advanced proteomics and metabolomics research. It combines a quadrupole, an ion trap, and an Orbitrap mass analyzer to provide high-performance mass analysis and tandem mass spectrometry (MS/MS) capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (9 mentions)
Sequencing grade modified porcine trypsin, by Promega (4 mentions)
Acclaim pepmap 100 c18 trap column, by Thermo Fisher Scientific (4 mentions)
Acclaim pepmap 100 c18 analytical column, by Thermo Fisher Scientific (3 mentions)
Dionex ultimate 3000 system, by Thermo Fisher Scientific (3 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (2 mentions)
Vanquish neo uhplc system, by Thermo Fisher Scientific (2 mentions)
Xselect csh c18 2.5 m resin, by Waters Corporation (2 mentions)
Ultimate 3000 rslcnano lc system, by Thermo Fisher Scientific (2 mentions)
Easy nlc 1200, by Thermo Fisher Scientific (2 mentions)
Acquity beh c18 column, by Waters Corporation (2 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (2 mentions)
Faims pro interface, by Thermo Fisher Scientific (2 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (2 mentions)
Easy nlc 1200 system, by Thermo Fisher Scientific (2 mentions)
Agilent 1290 infinity 2 uhplc system, by Agilent Technologies (1 mentions)
Proteome discoverer 2.4 sp1, by Thermo Fisher Scientific (1 mentions)
Pierce quantitative fluorometric peptide assay, by Thermo Fisher Scientific (1 mentions)
Proxeon easy nlc 1200 lc pump, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion lumos mass spectrometry, by Thermo Fisher Scientific (1 mentions)
38 protocols using orbitrap eclipse tribrid mass spectrometer
1
Targeted Proteomics by LC-MS/MS
2
HDX Analysis of CFTR Conformational Changes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Before the HDX experiments, P67L-CFTR-6SS was pre-incubated at 25 °C for 5 min in the absence or presence of 30 µM VX-809 or VX-445 correctors. DMSO (0.1%) was included in all conditions. HDX was initiated by mixing pre-incubated P67-CFTR-6SS into D2O-based buffer pre-incubated at 37 °C, at 1:9 dilution. The corrector concentration was kept at 30 µM during HDX incubation for 10 and 240 s at 37 °C. The HDX reaction was quenched by adding a 10 μl aliquot of the mixture into 5 μl of chilled quenching buffer (1 M glycine-HCl including 0.02% DDM, pH 2.4). Quenched samples were stored at −80 °C. The on-line pepsin digestion was carried at 60 μl/min flow rate for 1.5 min at 15 °C, and desalting was performed at a 200 μl/min flow rate for 1.5 min. Digestion mixtures were separated by an Agilent 1290 Infinity II UHPLC system using a 5–40% liner gradient of ACN containing 0.1% FA for 11 min at 65 μl/min. MS measurements were performed using an Orbitrap Eclipse tribrid mass spectrometer (ThermoFisher Scientific). Mass spectra of peptides were acquired in positive-ion mode for m/z 200–2000. Data analysis was carried out using HDExaminer 3.3 (Sierra Analytics). MS/MS spectra were analyzed using Proteome Discoverer 2.4 SP1 (ThermoFisher Scientific). All HDX data collected are included in Source Data file.
3
Peptide Preparation and Proteomic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were prepared using the Thermo EasyPep Mini MS Sample Prep Kit (Thermo Scientific, A4006) according to the manufacturer’s instructions. The dried peptides were then dissolved in 0.1% formic acid (Thermo Scientific, 5178) solution, and peptide concentration was tested using the Pierce Quantitative Fluorometric Peptide Assay (Thermo Scientific, 23290). LC-MS/MS experiments were performed on an EASY-nLC 1000 (Thermo Fisher Scientific, Waltham, MA, USA) connected to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). Proteomic analysis was performed using Proteome Discoverer 2.4 (Thermo Scientific) software, the Uniprot human database and SequestHT with Percolator validation. The detailed information for LC/MS processing and data analysis was described previously [59 (link)].
4
Micro-flow LC-MS/MS for Peptide Separation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Micro-flow liquid chromatography tandem mass spectrometry was performed on a Vanquish Neo UHPLC system (Thermo Fisher Scientific) coupled online to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific) operating in positive ion mode as previously described [25 (link)]. Briefly, samples were loaded directly onto the Acclaim PepMap 100 C18 column (2 μm particle size, 1 mm ID × 150 mm). The peptide mixture was separated at a flow rate of 50 μL/min using a linear gradient of acetonitrile from 3 to 28% (v/v), formic acid 0.1% (v/v) and 3% (v/v) DMSO and at a column temperature of 55 °C for 60 min. The eluting peptides were directly sprayed into the heated electrospray ionization (HESI) source of the mass spectrometer. Tandem mass spectra were acquired in DDA mode. From each MS scan, precursors were targeted for MS/MS scans if the charge was between 2 and 6 and the intensity exceeded 1e4. Fragmentation of the peptides was performed by higher-energy collision-induced dissociation (HCD).
5
High-Resolution LC-MS Proteomic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LC-MS was carried out on an EASY-nLC 1000 (Thermo Fisher Scientific) coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). Native peptide fractions were resuspended in 15 µl of 2% ACN and 0.2% formic acid, and 5 µl peptides per concatenated sample were loaded onto a monolithic column (Capillary EX-Nano MonoCap C18 HighResolution 2000, 0.1 × 2,000 mm; Merck) fitted with a silica-coated PicoTip emitter (New Objective FS360-20-10-D) and separated over 180 min at a flow rate of 500 nl/min with the following gradient: 2–6% solvent B (10 min), 6–40% B (140 min), 40–98% B (1 min), and 98% B (29 min). MS1 spectra were acquired in the Orbitrap at 120-K resolution with a scan range from 375 to 2,000 m/z, an AGC target of 4e5, and a maximum injection rate of 50 ms in Profile mode. Features were filtered for monoisotopic peaks with a charge state of 2–7 and a minimum intensity of 2.5e4, with dynamic exclusion set to exclude features after 1 time for 60 s with a 5-ppm mass tolerance. HCD fragmentation was performed with collision energy of 32% after quadrupole isolation of features using an isolation window of 0.7 m/z, an AGC target of 5e4, and a maximum injection time of 86 ms. MS2 scans were then acquired in the Orbitrap at 50-K resolution in centroid mode with the first mass fixed at 110. Cycle time was set at 1 s.
6
Comprehensive Mass Spectrometry Proteomic Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were reduced, alkylated, and purified by chloroform/methanol extraction prior to digestion with sequencing grade modified porcine trypsin (Promega, Madison, WI). Tryptic peptides were then separated by reverse-phase XSelect CSH C18 2.5 µm resin (Waters) on an in-line 150 × 0.075 mm column using an UltiMate 3000 RSLCnano system (Thermo). Peptides were eluted using a 90 min gradient from 97:3 to 60:40 buffer A:B ratio (Buffer A = 0.1% formic acid, 0.5% acetonitrile; Buffer B = 0.1% formic acid, 99.9% acetonitrile). Eluted peptides were ionized by electrospray (2.15 kV) followed by mass spectrometric (MS) analysis on an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA). MS data were acquired using the FTMS analyzer in profile mode at a resolution of 120,000 over a range of 375 to 1200 m/z. Following HCD activation, MS/MS data were acquired using the ion trap analyzer in centroid mode and normal mass range with a normalized collision energy of 30%.
7
Orbitrap-Based Proteomics Workflow
8
LC-MS/MS Characterization of Tryptic Peptides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In preparation for the liquid chromatography (LC)–tandem MS (MS/MS) analysis the lyophilized tryptic peptides were reconstituted with 10% formic acid. The peptides were then separated by an UltiMate 3000 RSLCnano LC System (Thermo Fisher Scientific, Braunschweig, Germany) equipped with an Acclaim PepMap 100-C18 trap column (Thermo Fisher Scientific) and an Acclaim PepMap RSLC-C18 analytical column (Thermo Fisher Scientific). Mobile-phase solvents A and B consisted of 0.1% formic acid in water and acetonitrile, respectively. Trapping was performed at a flow rate of 10 μL/min for 5 min with 3% B and separation was performed at a passive split flow of 300 nL/min for 95 min with 8% to 50% B over 51 min, 50% to 99% B over 14 min, 99% B for 15 min, and 99% to 3% B over 10 min. The eluted peptides were electrosprayed at a 2.0 kV spray and analyzed by an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled to the LC system. The MS analyses were operated in data-dependent acquisition (DDA) mode using higher-energy collision dissociation (HCD) for MS/MS fragmentation.
9
Organic Synthesis Characterization Techniques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents and solvents purchased from commercial sources were used without further purification. Nuclear magnetic resonance (NMR) solvents were purchased from Cambridge Isotope Laboratories Inc. All reactions were monitored under UV light (254 nm) using a thin layer chromatography on pre-coated silica gel glass plates from Merck. Flash column chromatography was performed using silica gel (Kieselgel 60 Art. 9385, 230–400 mesh) from Merck. 1H and 13C NMR spectra were recorded on Bruker 400 MHz FT-NMR. Chemical shifts are reported in parts per million (ppm, δ) using peaks from an NMR solvent (CDCl3, CD3OD, or DMSO-d6) as a reference. Coupling constants (J) are reported in Hertz (Hz), and the multiplicities of peaks are abbreviated as s: singlet, br: broad singlet, d: doublet, t: triplet, q: quartet, dd: doublet of doublet, dt: doublet of triplet, and m: multiplet. High-resolution mass-spectral results were obtained using Orbitrap Eclipse™ Tribrid™ Mass Spectrometer (ThermoFisher Scientific, Waltham, MA, USA).
10
Orbitrap DIA Mass Spectrometry Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1 µl of sample was analysed on an Orbitrap Eclipse Tribrid Mass Spectrometer (Thermo Fisher) equipped with a nanoelectrospray source, connected to a nano-flow LC system (Easy-nLC 1200, Thermo Fisher). Peptides were separated on a 40 cm × 0.75 μm (inner diameter) column packed in-house with 1.9 μm C18 beads at a flow rate of 300 nl/min and a 60-min linear gradient from 3% to 30% II (Eluent I: 0.1% FA, Eluent II: 95% ACN, 0.1% FA). The column was heated to 50°C. The sample was measured three times in data-independent acquisition (DIA) mode with 41 variable width DIA windows with a 1 m/z overlap. For survey MS1 spectra the measured mass range was 350–1400 m/z at a resolution of 120,000 with 200% normalized AGC target or 100ms maximum injection time. Survey MS1 spectra were acquired in between complete DIA isolation window sets. MS2 spectra covered a mass range of 150–2000 m/z at a resolution of 30,000. HCD collision energy for MS2 spectra was set to 30% with 400% normalized AGC target or 54-ms maximum injection time.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!