From each of the 6 fractions, ~5 μg was dissolved in 15% aqueous formic acid/5% acetonitrile prior to LC-MS/MS analysis on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to a Proxeon EASY-nLC 1200 liquid chromatography (LC) pump (Thermo Fisher Scientific). Peptides were fractionated on a 100-μm inner diameter microcapillary column packed with ~35 cm of Accucore resin (2.6 μm, 150 Å, ThermoFisher Scientific). For each analysis, we loaded ~1 μg onto the column. Subsequent separation and acquisition were performed as previously described5 (link),14 . Samples were analyzed in duplicate, one with advanced peak determination (ADP) activated and a second run with this option off. Both analyses used the real-time search (RTS) algorithm67 (link). Data was searched against the UniProt human database (downloaded: October, 2016).
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Quantitative Proteomics via LC-MS/MS
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Corresponding Organization : University of Bergen
Other organizations : University of Oslo, Oslo University Hospital, Harvard University, Haukeland University Hospital
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Variable analysis
independent variables
- Activation of advanced peak determination (ADP)
- Use of real-time search (RTS) algorithm
- Peptide separation and acquisition
- Concentration of sample loaded onto the column (~1 μg)
- Solvent composition (15% aqueous formic acid/5% acetonitrile)
- LC-MS/MS platform (Orbitrap Fusion Lumos mass spectrometer coupled to Proxeon EASY-nLC 1200 liquid chromatography pump)
- Column specifications (100-μm inner diameter microcapillary column packed with ~35 cm of Accucore resin, 2.6 μm, 150 Å)
- Data search against the UniProt human database (downloaded: October, 2016)
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