Profiling of the mussel extracts was performed as previously described for secretome samples (41 (link)). Briefly, 100 µg of crude and clarified mussel extracts were enzymatically digested using a trypsin/Lys-C mixture, followed by desalting and purification using STop And Go Extraction (STAGE)-tips (42 (link)). Samples were measured on an Orbitrap Exploris 240 hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Easy-nLC 1200 high-performance liquid chromatography device (Thermo Fisher Scientific). Samples were loaded onto an in-line 75 mm by 50 cm PepMap RSLC EASY-Spray column filled with 2-mm C18 reverse-phase silica beads (Thermo Fisher Scientific). Separated peptides were electro-sprayed into the mass spectrometer with a linear gradient of 3–20% buffer B (80% acetonitrile and 0.5% acetic acid) over a 60-min gradient, followed by a wash with 100% buffer B with a 250 nL/min flow rate. The mass spectrometer was operated in a data-dependent acquisition model and switched between one full scan and MS/MS scans of abundant peaks. Full scans (m/z 400 to 2,000) were acquired in the Orbitrap mass analyzer with a resolution of 120,000 at m/z 200.
2 mm c18 reverse phase silica beads
Manufactured by Thermo Fisher Scientific
The 2-mm C18 reverse-phase silica beads are a type of chromatographic media used in analytical and preparative liquid chromatography. These beads have a particle size of 2 millimeters and a stationary phase consisting of C18 (octadecyl) hydrocarbon chains bonded to a silica support. The C18 groups provide hydrophobic interaction for the separation and purification of a wide range of organic compounds.
Automatically generated - may contain errors
2 protocols using 2 mm c18 reverse phase silica beads
1
Mass Spectrometry Profiling of Mussel Extracts
2
High-resolution Proteomics Workflow with Orbitrap
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Samples were run over a 60-min chromatographic gradient with an Orbitrap Exploris 240 hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Easy-nLC 1200 high-performance liquid chromatography device (Thermo Fisher Scientific). Samples were loaded onto an in-line 75-mm by 50-cm PepMap RSLC EASY-Spray column filled with 2-mm C18 reverse-phase silica beads (Thermo Fisher Scientific). Separated peptides were electro-sprayed into the mass spectrometer with a linear gradient of 3% to 20% buffer B (80% acetonitrile, 0.5% acetic acid) over a 3 h gradient, followed by a wash with 100% buffer B with a 250-nL/min flow rate. The mass spectrometer switched between one full scan and MS/MS scans of abundant peaks. Full scans (m/z 400 to 2,000) were acquired in the Orbitrap mass analyzer with a resolution of 120,000 at m/z 200.
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