Magic c18 aq 3 μm

The setup of the μRPLC-MS system was as described previously [23 (link)]. The hybrid LTQ-Orbitrap XL mass spectrometer was interfaced to a nanoelectrospray ion source (both Thermo Electron, Bremen, Germany) coupled online to a Tempo 1D-plus nanoLC (Applied Biosystems/MDS Sciex, Foster City, CA). 1 μg of total peptide mass was separated on a RP-LC column (75 μm × 15 cm) packed in-house with C18 resin (Magic C18 AQ 3 μm; Michrom BioResources, Auburn, CA, USA) using a linear gradient from 96% solvent A (98% water, 2% acetonitrile, 0.15% formic acid) and 4% solvent B (98% acetonitrile, 2% water, 0.15% formic acid) to 30% solvent B over 120 minutes at a flow rate of 0.3 μl/min. Each survey scan acquired at 60,000 FWHM was followed by MS/MS scans of the five most intense precursor ions in the linear ion trap with dynamic exclusion enabled for a period of 60 seconds. Charge state screening was employed to select for ions with at least two charges and rejecting ions with undetermined charge state. The normalized collision energy was set to 32% and one microscan was acquired for each spectrum.

Samples were centrifuged at 14,000–16,000 × g for 10 min to remove particulate material. Five microliters of each sample were injected into a self-packed fused silica nano column, which had been pulled to a 5 μm i.d. tip using a P-2000 CO₂ laser puller (Sutter Instruments, Novato, CA), then packed with 13 cm of 3 μm C18 reverse phase (RP) particles (Magic C18 AQ 3 μm (Michrom. Bioresources, Auburn, CA)) and equilibrated in 5 % acetonitrile, 0.1 % formic acid. Full MS spectra were recorded on the peptides over a 400 to 2000 m/z range by the Orbitrap, followed by five tandem mass (MS/MS) events sequentially generated by LTQ in a data-dependent manner on the first, second, third, and fourth most intense ions selected from the full MS spectrum (at 35 % collision energy). Mass spectrometer scan functions and HPLC solvent gradients were controlled by the Xcalibur data system (Thermo Finnigan, San Jose, CA).

Prior to mass spectrometry analysis of RIPK1 interactors, eluted protein complexes were digested with Trypsin and peptides were purified using C18 Microspin columns (Harvard Apparatus) according to the manufactures instruction. LC-MS/MS analysis was performed on a dual pressure LTQ-Orbitrap mass spectrometer (Thermo Scientific), which was connected to an electrospray ion source (Thermo Scientific). Peptide separation was carried out using an easy nano-LC systems (Proxeon Biosystems) equipped with an RP-HPLC column packed with C18 resin (Magic C18 AQ 3 μm; Michrom BioResources). A 0.3 μl/min linear gradient from 96% solvent A (0.15% formic acid, 2% acetonitrile) and 4% solvent B (98% acetonitrile, 0.15% formic acid) to 40% solvent B over 40 min. The data acquisition mode was set to obtain one high-resolution MS scan in the FT part of the mass spectrometer at a resolution of 60,000 FWHM followed by MS/MS scans in the linear ion trap of the 20 most intense ions. Raw files were converted to the mzXML format, and searched against the human swissprot protein database. Further data processing including SAINT was carried out as described previously (Choi et al., 2011 (link)).