Ultimate nanorslc uplc and autosampler system

TiO₂ and Fe-NTA phosphopeptide enriched samples were each resuspended in 10 µl of 1% formic acid, 0.05% heptafluorobutyric acid and 2% acetonitrile. The samples (2.5 - 5 µl) were then loaded onto the Fusion Lumos (ThermoFisher) connected to an UltiMate nanoRSLC UPLC and autosampler system (Dionex).
The peptides were initially concentrated and desalted with H2O:CH3CN (98:2, 0.2% TFA) at 15 µl/min on a micro C18 precolumn (300 µm x 5 mm, Dionex). After a 4 min wash, the micro C18 precolumn was switched (Valco 10 port UPLC valve, Valco) into line to a fritless nano column (75µ x ~15cm), which contained C18AQ media (1.9µ, 120 Å Dr Maisch). Peptides were then separated on a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 0.2 µl/min over 30 min. Positive ions were generated with electrospray ionization at 2000V. Data dependent acquisition (DDA) was performed with survey scan from m/z of 350 to 1750, resolution of 120,000 at m/z 200, accumulation target value of 400,000 ions and lockmass enabled (m/z 445.12003). A top-speed approach with a cycle time of 2s was used for data-dependent tandem MS analysis. Ions were fragmented by higher-energy collisional dissociation (HCD) with intensity threshold at 25,000. A mass tolerance of 10 ppm and dynamic exclusion of 20s was set.

Samples were reduced (5 mM DTT, 37 °C, 30 min), alkylated (10 mM IA, RT, 30 min), and incubated with trypsin at 37 °C for 18 h, at a 1:20 ratio (w/w). Samples were desalted with two SDB-RPS disks (Empore, Sigma Cat # 66886-U) packed in the 200 µL pipette tip as described previously [77 (link)]. Eluted peptides from each clean-up were reconstituted in 10 µL 0.1% (v/v) formic acid and 0.05% (v/v) heptafluorobutyric acid in water. Proteolytic peptide samples were separated by nanoLC using an Ultimate nanoRSLC UPLC and autosampler system (Dionex, Amsterdam, The Netherlands) and analyzed on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific, Bremen, Germany), as described previously [78 (link)]. A 90 min gradient was used.

For the mass spectrometry analysis, total protein was determined by homogenising the cells in RIPA buffer (Life Technologies, Carlsbad, CA, USA) containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Samples were sonicated using the Bioruptor Pico (Diagenode, Liège, Belgium) for a total of 10 min. Protein concentrations were determined using the 2-D Quant kit (Cytiva Life Sciences, Marlborough, MA, USA). Samples were reduced (5 mM DTT, 37C, 30 min), alkylated (10 mM IA, RT, 30 min) then incubated with trypsin at a protease:protein ratio of 1:20 (w/w) at 37 °C for 18 h, before being subjected to SCX clean-ups (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. Eluted peptides from each clean-up were evaporated to dryness in a SpeedVac (Thermo Fisher Scientific, Waltham, MA, USA) and reconstituted in 20 µL 0.1% (v/v) formic acid. Proteolytic peptide samples were separated by nanoLC using an Ultimate nanoRSLC UPLC and autosampler system (Dionex, Sunnyvale, CA, USA) and analysed on a Tribrid Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) as described before [27 (link)].