C18 particles

The proteins bound to streptavidin beads were reduced and alkylated via sequential 20 minute incubations of 5mM TCEP 10mM iodoacetamide at room temperature in the dark while being mixed at 1200 rpm an Eppendorf thermomixer. Proteins were then digested by the addition of 0.1μg Lys-C (FUJIFILM Wako Pure Chemical Corporation, 125–05061) and 0.8μg Trypsin (Thermo Scientific, 90057) while shaking 37°C overnight. The digestions were quenched via addition of formic acid to a final concentration of 5% by volume. Each sample was desalted via C18 tips (Thermo Scientific, 87784) and then resuspended in 15μL of 5% formic acid before analysis by LC-MS/MS. Peptide samples were then separated on a 75μM ID, 25cm C18 column packed with 1.9μM C18 particles (Dr. Maisch GmbH) using on a 140-minute gradient of increasing acetonitrile and eluted directly into a Thermo Orbitrap Fusion Lumos instrument where MS/MS spectra were acquired by Data Dependent Acquisition (DDA). Data analysis was performed using the ProLuCID and DTASelect2 algorithms as implemented in the Integrated Proteomics Pipeline—IP2 (Integrated Proteomics Applications, Inc., San Diego, CA). Protein and peptide identifications were filtered using DTASelect and required a minimum of two unique peptides per protein and a peptide-level false positive rate of less than 1% as estimated by a decoy database strategy.

Peptides were separated on an EASY-nLC 1000 HPLC system (Thermo Fisher Scientific) via in-house packed columns (75 μm inner diameter, 50 cm length, and 1.9 μm C18 particles [Dr. Maisch GmbH]) in a gradient of buffer A (0.5% formic acid) to buffer B (80% acetonitrile, 0.5% formic acid). The gradient started at 5% B, increasing to 30% B in 65 min, further to 95% B in 10 min, staying at 95% B for 5 min, decreasing to 5% B in 5 min, and staying at 5% B for 5 min at a flow rate of 300 nl/min and a temperature of 60 °C. A Quadrupole Orbitrap mass spectrometer (Q Exactive HF-X; Thermo Fisher Scientific) was directly coupled to the LC via a nano-electrospray source. The Q Exactive HF-x was operated in a data-dependent mode. The survey scan range was set from 300 to 1650 m/z, with a resolution of 60,000 at m/z 200. Up to the 12 most abundant isotope patterns with a charge of two to five were isolated and subjected to collision-induced dissociation fragmentation at a normalized collision energy of 27, an isolation window of 1.4 Th, and a MS/MS resolution of 15,000 at m/z 200. Dynamic exclusion to minimize resequencing was set to 30 s.

Analysis of the peptide mixtures was performed as described previously [25 (link)]. Briefly, the peptides were fractionated on a reversed phase column (50 cm × 75 μm inner diameter) packed with 1.8 μm diameter C18 particles (100 Å pore size; Dr. Maisch, Ammerbuch-Entringen, Germany) using a 105 min acetonitrile gradient in 0.1% formic acid at a flow rate of 250 nL/min. Peptide masses were analyzed using a Q-Exactive HF mass spectrometer (Thermo-Fisher Scientific, Palo Alto, CA, USA) operated in data-dependent mode with survey scans acquired at a resolution of 50,000 at m/z 400 (transient time 256 ms). Fifteen of the most abundant isotope patterns with charge ≥ +2 from the survey scan (300–1650 m/z) were selected with an isolation window of 1.6 m/z and fragmented by HCD with normalized collision energies of 25. The maximum ion injection times for the survey scan and the MS/MS scans were 20 and 60 ms, respectively. The ion target values for MS1 and MS2 scan modes were set to 3 × 10⁶ and 1 × 10⁵, respectively. The dynamic exclusion was 25 s and 10 ppm.