Gel bands were cut into 1 mm3 pieces and were subjected to in-gel trypsin digestion overnight. The peptides were extracted with acetonitrile and vacuum dried. Samples were loaded onto Proxeon EASY-nLC II liquid chromatography pump (Thermo Fisher) after reconstituted in HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). By increasing the concentration of solvent B (97.5% acetonitrile, 0.1% formic acid), samples were eluted with a gradient of acetonitrile (6–30%) within 30 min. The eluates were directly subjected to Orbitrap Elite MS (Thermo Fisher). To produce a tandem mass spectrum of specific fragment ions for each peptide, the peptides were detected, isolated, and fragmented. The MS/MS spectra were analyzed by matching protein databases with the acquired fragmentation patterns using SEQUEST (ver. 28, Thermo Fisher). Enzyme specificity was set to partially tryptic with two missed cleavages. Carboxyamidomethyl for cysteine and oxidation for methionine residues were set as static modifications and variable modification respectively. According to the target-bait method, the identified peptides were filtered with false discovery rate (FDR) < 1%. A complete list of peptides identified by mass spectrometry was shown in Table S1 .
Orbitrap elite ms
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United Kingdom
The Orbitrap Elite MS is a high-performance mass spectrometer that utilizes Orbitrap technology to provide accurate and resolute mass analysis. It is designed to deliver exceptional performance, sensitivity, and versatility for a wide range of applications in analytical chemistry and life sciences.
Automatically generated - may contain errors
Lab products found in correlation
Proxeon easy nlc 2, by Thermo Fisher Scientific (1 mentions)
Avance 3 400 spectrometer, by Bruker (1 mentions)
Agilent 7890b, by Agilent Technologies (1 mentions)
Instantblue, by Abcam (1 mentions)
Ultimate 3000 rapid separation lc, by Thermo Fisher Scientific (1 mentions)
Bridged ethyl hybrid c18 analytical column, by Waters Corporation (1 mentions)
Hypersil gold reversed phase c18 column, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 uhplc system, by Thermo Fisher Scientific (1 mentions)
Xcalibur, by Thermo Fisher Scientific (1 mentions)
Negative ion calibration solution, by Thermo Fisher Scientific (1 mentions)
9 protocols using orbitrap elite ms
1
Comprehensive Proteomic Workflow for Peptide Analysis
2
Peptide Identification by Orbitrap MS/MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The excised bands were separately subjected to in-gel trypsin digestion and the resulting peptides solubilized in 20-μL 5% acetonitrile with 0.5% formic acid using the auto-sampler of a nanoflow uHPLC system (Thermo Scientific RSLCnano). Online detection of peptide ions was by electrospray ionization-mass spectrometry MS/MS with an Orbitrap Elite MS (Thermo Scientific). Data were submitted for an MS/MS ion search via the Mascot search engine (http://www.matrixscience.com ).
3
Synthesis and Characterization of Organic Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals, reagents and solvents were purchased from commercial sources. When necessary, they were purified and dried by standard methods. Reactions were checked by thin-layer chromatography (TLC) on pre-coated silica gel F254 plates. Column chromatography was carried out with Silica gel H (200–300 mesh or 500 mesh). Detection was by iodine vapour staining and UV light irradiation (UV lamp, model UV-IIB). Melting points were determined on an X4-type apparatus and are not corrected. 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE III-400 spectrometer, Chemical shifts δ in ppm with Me4Si as internal standard, coupling constants J in Hertz. High-resolution mass spectrum (HRMS) was recorded on a Thermo Scientific Orbitrap Elite MS.
4
High-Resolution Orbitrap Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All mass spectra were collected on a research‐type Orbitrap Elite MS (Thermo Scientific, Bremen, Germany), using an electrospray ionization source in positive mode [ESI(+)] at 4 kV ionization voltage. Mass spectra were recorded over a range of 150≤m/z≤1000 with a resolution setting of R=480000 (full width at half‐maximum at m/z=400). Spectral stitching was used with scan windows of 30 Da and a 5 Da overlap.[20 , 23 ]
GC‐FID measurements were conducted on an Agilent 7890B with a DB‐Wax‐Ether column (Agilent) (L=30 m, I.D.=0.25 mm). The carrier gas was H2 at 0.6 bar, and the temperature was increased from 35 to 280 °C at 5 °C min−1 and held at 280 °C for 5 min. The injector had a temperature of 220 °C, the analyzer one of 350 °C.
GC‐FID measurements were conducted on an Agilent 7890B with a DB‐Wax‐Ether column (Agilent) (L=30 m, I.D.=0.25 mm). The carrier gas was H2 at 0.6 bar, and the temperature was increased from 35 to 280 °C at 5 °C min−1 and held at 280 °C for 5 min. The injector had a temperature of 220 °C, the analyzer one of 350 °C.
5
Identification of Talin-1 Phosphorylation Sites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phosphorylation sites of talin-1 R7R8 were determined using mass spectrometry. The in vitro kinase assay was carried out with the substrate talin-1 R7R8 and CDK1-cyclin A2. Reactions was carried out for 45 min at 30 °C and stopped by adding SDS sample buffer and boiling for 10 min at 95 °C. All samples were loaded onto an SDS PAGE 4 to 12% Bis-Tris gel (Thermo Fisher Scientific) and separated by running at 200 V for 60 min. Gels were stained with Instant-Blue (Expedeon) for 15 min and washed in water overnight at 4 °C. The talin R7R8 bands were cut from the gel and processed by in-gel tryptic digestion. Peptides were analyzed by LC-MS/MS by using an UltiMate 3000 Rapid Separation LC (Dionex Corporation) coupled to an Orbitrap Elite MS (Thermo Fisher Scientific). Peptides were separated on a bridged ethyl hybrid C18 analytical column (250 mm × 75 μm internal diameter, 1.7 μm particle size; Waters) over a 45 min gradient from 8 to 33% (v/v) acetonitrile in 0.1% (v/v) formic acid. LC-MS/MS analyses were operated in data-dependent mode to automatically select peptides for fragmentation by CID. Multistage activation was enabled to fragment product ions resulting from neutral loss of phosphoric acid. Quantification was performed using Progenesis LC-MS/MS software.
6
Orbitrap-based Metal-Ligand Complex Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Metal–ligand complex analysis in an ion trap MS instrument involved a high-resolution electrospray ionization (ESI)-coupled Orbitrap Elite Mass spectrometer (Thermo Scientific Orbitrap Elite MS). In direct infusion ESI-MS analysis, 10 μL analyte samples was injected into the instrument by loop (Waters) injection and delivered to the ionization source (Heated-ESI, HESI) with subsequent injection of 10 μL of 0.1% formic acid (FA) in 80% acetonitrile (ACN). The Orbitrap mass analyzer was operated in positive ionization mode with a source voltage 2.5 KV, capillary temperature 360 °C and source heater temperature 350 °C. Data were acquired at resolution 15,000 with a Fourier transform (FT)-MS instrument in a range of mass-to-charge ratios (m/z) of 50–990 and isolation width of 2 Thomson. Tandem mass spectra (MS/MS; MS2) acquired with high-energy collisional dissociation (HCD) were produced at different collision energies, 40–150%, and 2.0 ppm tolerance window. Data were analyzed by use of Thermo Scientific Xcalibur software (Thermo Fishers).
7
Photolabeling and Top-Down Mass Spectrometry of mVDAC1
8
Targeted Metabolomics Analysis by UHPLC-Orbitrap
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analysis was performed on a Dionex Ultimate 3000 UHPLC system (Dionex, now Thermo Fisher Scientific, Hemel Hempstead, UK) interfaced via an electrospray ionisation (ESI) probe to an Orbitrap Elite MS (Thermo Fisher Scientific). Chromatographic separation was carried out on a Hypersil Gold reversed phase C18 column (1.9 µm particle size, 50 x 2.1 mm, Thermo Fisher Scientific, UK). Details of the mobile phase and gradients employed are given in Supplemental Materials and Methods. MS analysis on the Orbitrap Elite was performed in the positive-ion mode with five scan events, one high resolution (120,000 full width at half maximum height at m/z 400) scan over the m/z range 400 – 610 in the Orbitrap and four MS3 scans performed in parallel in the linear ion trap (LIT). Mass accuracy in the Orbitrap was typically < 5 ppm. More details of the scan events are provided in Supplemental Materials and Methods. Injection volumes were 35 µL for plasma extracts and at 90 µL for amniotic fluid and placental extracts.
9
Myo-IP6 Identification via HRMS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reference myo-IP6 solution was prepared at 20 ppm in DDW. Preparatory IC eluate was analysed directly. HRMS analysis was performed on an Orbitrap Elite MS (Thermo Scientific)
with an ESI source. The Orbitrap was operated in negative ion mode, calibrated using negative ion calibration solution (Thermo Scientific), and tuned automatically on the m/z 328.9 (myo-
). Solutions were directly infused at 10 µL min -1 for acquisition of full mass spectra. Source voltage was ˗1.8 kV, sheath gas (nitrogen) flow rate was 30 arbitrary units (arb), auxiliary gas (nitrogen) flow rate was 0 arb and the sweep gas (nitrogen) flow rate was 1 arb. The capillary temperature was set to 275 ° C. Full mass spectra were recorded at 120,000 resolution and 50 scans were averaged to increase the signal-to-noise ratio. Mass spectra were analysed using Xcalibur (Thermo Scientific). Myo-IP6 was identified by comparison of the negative ion mass spectrum of the eluate to that of reference myo-IP6 as described in McIntyre et al. [27] (link) and observation of characteristic ions with m/z values < 5 ppm of those reported for the standard compound.
with an ESI source. The Orbitrap was operated in negative ion mode, calibrated using negative ion calibration solution (Thermo Scientific), and tuned automatically on the m/z 328.9 (myo-
). Solutions were directly infused at 10 µL min -1 for acquisition of full mass spectra. Source voltage was ˗1.8 kV, sheath gas (nitrogen) flow rate was 30 arbitrary units (arb), auxiliary gas (nitrogen) flow rate was 0 arb and the sweep gas (nitrogen) flow rate was 1 arb. The capillary temperature was set to 275 ° C. Full mass spectra were recorded at 120,000 resolution and 50 scans were averaged to increase the signal-to-noise ratio. Mass spectra were analysed using Xcalibur (Thermo Scientific). Myo-IP6 was identified by comparison of the negative ion mass spectrum of the eluate to that of reference myo-IP6 as described in McIntyre et al. [27] (link) and observation of characteristic ions with m/z values < 5 ppm of those reported for the standard compound.
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!