The lyophilized peptides were dissolved in 2% acetonitrile with 0.5% formic acid for nano LC-ESI-MS/MS analysis, which was carried out on a LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The Orbitrap was interfaced with Dionex UltiMate3000 MDLC system (Thermo Dionex, Sunnyvale, CA). Protein samples were injected onto a Acclaim PepMap nano Viper C18 trap column (5 µm, 100 µm × 2 cm, Thermo Dionex) at a flow rate of 20 µL/min for on-line desalting and then separated on C18 RP nano column (5 µm, 75 µm × 50 cm, Magic C18, Bruker). The gradient for HPLC condition was 5–38% acetonitrile with 0.1% formic acid in 120 min. The flow rate was 0.3 µL/min. The Orbitrap Elite was operated in positive ion mode with spray voltage 1.6 kV and source temperature 275 °C. Data-dependent acquisition (DDA) mode was used by one precursor ions MS survey scan from m/z 300 to 1800 at resolution 60,000 using FT mass analyzer, followed by up to 10 MS/MS scans at resolution 15,000 on 10 most intensive peaks. All data were acquired in Xcalibur 2.2 operation software (Thermo Fisher Scientific).
Xcalibur 2.2 operation software
Manufactured by Thermo Fisher Scientific
Xcalibur 2.2 is an operation software for analytical instrumentation. It provides a user interface for instrument control, data acquisition, and data processing. The software supports a range of analytical techniques and is compatible with various Thermo Fisher Scientific instrument platforms.
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2 protocols using xcalibur 2.2 operation software
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Nano LC-ESI-MS/MS Analysis of Lyophilized Peptides
2
Nano LC-ESI-MS/MS Analysis of Lyophilized Peptides
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The lyophilized peptides were dissolved in 2% acetonitrile with 0.5% formic acid for nano LC-ESI-MS/MS analysis, which was carried out on a LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The Orbitrap was interfaced with Dionex UltiMate3000 MDLC system (Thermo Dionex, Sunnyvale, CA). Protein samples were injected onto a Acclaim PepMap nano Viper C18 trap column (5 µm, 100 µm × 2 cm, Thermo Dionex) at a flow rate of 20 µL/min for on-line desalting and then separated on C18 RP nano column (5 µm, 75 µm × 50 cm, Magic C18, Bruker). The gradient for HPLC condition was 5–38% acetonitrile with 0.1% formic acid in 120 min. The flow rate was 0.3 µL/min. The Orbitrap Elite was operated in positive ion mode with spray voltage 1.6 kV and source temperature 275 °C. Data-dependent acquisition (DDA) mode was used by one precursor ions MS survey scan from m/z 300 to 1800 at resolution 60,000 using FT mass analyzer, followed by up to 10 MS/MS scans at resolution 15,000 on 10 most intensive peaks. All data were acquired in Xcalibur 2.2 operation software (Thermo Fisher Scientific).
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