The lyophilized powders were reconstituted in 10 μL of 0.1% FA in H2O and analyzed by LTQ Orbitrap XL (Thermo Fisher Scientific, San Jose, CA). Reverse-phase nanoLC separation was performed on an Agilent 1200 series nanoflow system (Agilent Technologies, Santa Clara, CA). A total of 10 μL sample from collected fractions was loaded onto an Agilent Zorbax XDB C18 precolumn (0.35 mm, 5 μm), followed by separation using in-house C18 column (i.d. 75 μm × 15 cm, 3 μm). The mobile phases used were (A) 0.1% FA in water and (B) 0.1% FA in 100% ACN. A linear gradient from 5% to 95% of (B) over a 70-min period at a flow rate of 300 nL/min was applied. The peptides were analyzed in the positive ion mode by applying a voltage of 1.8 kV to the injection needle. The MS was operated in a data-dependent mode, in which one full scan with m/z 400–1600 in the Orbitrap using a scan rate of 30 ms/scan. The fragmentation was performed using the CID mode with collision energy of 35 V. A repeat duration of 30 s was applied to exclude the same m/z ions from the reselection for fragmentation. The Xcalibur software (version 2.0.7, Thermo Fisher Scientific, San Jose, CA) was used for the management of instrument control, data acquisition, and data processing.
Agilent 1200 series nanoflow system
Manufactured by Agilent Technologies
The Agilent 1200 series nanoflow system is a high-performance liquid chromatography (HPLC) system designed for ultra-low flow rate applications. It is capable of generating and controlling flow rates in the nanoliter per minute range, making it suitable for sensitive analytical techniques such as mass spectrometry.
Automatically generated - may contain errors
Lab products found in correlation
Ltq orbitrap xl, by Thermo Fisher Scientific (2 mentions)
Xcalibur software, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx precast gradient gel, by Bio-Rad (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Promega (1 mentions)
4 protocols using agilent 1200 series nanoflow system
1
Proteomic Analysis by Orbitrap Mass Spectrometry
2
Furin Cleavage Peptide Purification
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Furin cleavage reactions were set up as described above, except that BSA was not added to the reactions. The reaction mixtures were incubated for 60 min at room temperature and were stopped by adding EGTA to 10 mM concentration to inhibit furin. Reactions where 10 mM EGTA was present at the time of furin addition were carried out for controls. The peptides arising from furin cleavage were purified using C18 StageTips. The peptides were separated using Agilent 1200 series nanoflow system (Agilent Technologies) and sprayed into an LTQ Orbitrap mass spectrometer (Thermo Electron) with a nanoelectrospray ion source (Proxeon). The data was analyzed using MaxQuant49 (link).
3
Quantitative Proteomic Analysis of Reversed CB Samples
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Reversed CB samples were separated in mini-protean TGX pre-cast gradient gels (BioRad) and stained with SimplyBlue SafeStain (Life Technologies). Gel pieces were excised from the sample lanes, followed by in-gel digestion with trypsin (Promega) and extraction of the peptides. The peptides were analyzed using LC-MS/MS with an Agilent 1200 series nanoflow system (Agilent Technologies) connected to a LTQ Orbitrap mass-spectrometer (Thermo Electron) equipped with a nanoelectrospray ion source (Proxeon, Odense). The data were analyzed using the Mascot search engine and the NCBI MOUSE database. For the approximate quantitation, the exponentially modified Protein Abundance Index (emPAI) was used.
4
Nano-LC-MS/MS Analysis of Lyophilized Samples
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The lyophilized powders, reconstituted in 10 μL of 0.1% FA in H2O, were analyzed by LTQ Orbitrap XL (Thermo Fisher Scientific, San Jose, Calif). Reverse-phase nano-liquid chromatography (nano-LC) separation was performed on an Agilent 1200 series nanoflow system (Agilent Technologies, Santa Clara, Calif). A total of 10-μL sample from collected fractions was loaded onto an Agilent Zorbax XDB C18 precolumn (0.35 mm, 5 μm), followed by separation using in-house C18 column (ID 75 μm × 15 cm, 3 μm). The mobile phases used were A 0.1% FA in water and B 0.1% FA in 100% ACN. The linear gradient from 5% to 95% of B over a 70-minute period at a flow rate of 300 nL/min was applied. The peptides were detected in the positive ion mode by applying a voltage of 1.8 kV to the injection needle. The MS was operated in a data-dependent mode, in which 1 full scan with m/z 400 to 1600 in the Orbitrap using a scan rate of 30 ms/scan. The fragmentation was performed using the collision-induced dissociation mode with a collision energy of 35 V. A repeat duration of 30 seconds was applied to exclude the same m/z ions from being taken as the reselection for fragmentation. The Xcalibur software (version 2.0.7; Thermo Fisher Scientific, San Jose, Calif) was used for the management of instrument control, data acquisition, and data processing.
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