Pepmap rslc

The mass spectrometry experiments were carried out at the UNC Proteomics Core Facility. Bands corresponding to Raptor were in-gel digested with trypsin overnight. Extracted peptides were enriched for phosphopeptides using TiO₂. The TiO₂ elution for each sample was analyzed by LC/MS/MS on a ThermoScientific Q-Exactive HF mass spectrometer. Samples were fractionated by C18 (Thermo PepMap RSLC) over a 45 min gradient from 5–35%B, where mobile phase A = 0.1% formic acid and mobile phase B = acetonitrile with 0.1% formic acid (ThermoScientific Easy nLC 1000). The top 15 most intense ions were chosen for HCD fragmentation. Data were searched against a reviewed Human UniProt database using Mascot. The parameters used were: 10 ppm precursor ion mass tolerance, 0.02 Da product ion mass tolerance, up to two missed trypsin cleavage sites, carbamidomethylation of Cys was set as a fixed modification and oxidation of M, deamidation of N, Q, and phospho of S, T, Y were set as variable modifications. A peptide false discovery rate of 5% was used to filter all results.

Proteins were digested and analyzed as previously described in Turetschek et al. (2017) (link). The protein pellet was dissolved in urea buffer (8 M urea, 50 mM HEPES, pH 7.8) and quantified with Bradford assay. For each sample, 100 μg protein was digested with Lys-C (1:100 v/v, 5 h, 30°C, Roche, Mannheim, Germany) and trypsin (1:10, v/v, over- night, 37°C, Applied Biosystems, Darmstadt, Germany). The sample was acidified with 200 mm³ 8% formic acid (FA) and loaded on stage tips (Pierce^TM C18 Tips, 100 mm³). Peptides were washed 4 times with 200 mm³ 0.1% FA, eluted with 0.1% FA in methanol, split in two aliquots and stored at -80° in a protein LoBind tube until measurement.
Peptides of 4 biological replicates were dissolved in 100 mm³ 2% ACN, 0.1% FA. In random order 1 μg was applied on a C18 column (15 cm × 50 μm column, PepMap^®RSLC, Thermo Scientific, 2 μm particle size) for separation during a 110 min gradient at a flow rate of 300 μm³ min^-1. Measurement was done on an LTQ-Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) with following settings: Full scan range 350–1800 m/z, max. 20 MS2 scans (activation type CID), repeat count 1, repeat duration 30 s, exclusion list size 500, exclusion duration 60 s, charge state screening enabled with rejection of unassigned and +1 charge states, minimum signal threshold 500.

One microgram of desalted and dried peptide samples were dissolved in 2% ACN and 0.1% formic acid (FA). Two technical replicates per sample were injected in random order to an EASY-Spray column, 15 cm × 50 μm ID, PepMap C18, >2 μm particles, 100 Å pore size (PepMap RSLC, Thermo Scientific) and separated during a 180 min gradient with a flow rate of 400 nL/min using an 1D nano LC (UltiMate 3000, Thermo Fisher Scientific) coupled to an Orbitrap Elite Hybrid Ion Trap-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) with full scan range 350–1,800 m/z, enabled dynamic exclusion, exclusion duration 60 s, exclusion list size 500, repeat duration 30 s, repeat count 1, 20 MS2 scans, CID (activation type), enabled charge state rejection with rejected unassigned charge state 1, minimum required signal 1,000 and expiration count 1.

Samples were reconstituted in 0.3% formic acid and peptide concentrations were measured using a NanoDrop spectrometer. 2.5 µg of tryptic peptides and 1 µg of deglycosylated peptides were analyzed in each LC-MS/MS run on an Orbitrap Fusion (Thermo Scientific) coupled to a Dionex Ultimate 3000 (Thermo Scientific) via a nanoelectrospray ion source. Samples were loaded on a 2 cm C18 trap column (Acclaim PepMap100, Thermo Scientific) and separated using a 2.5 h (90 min for S. aureus stimulation experiments) non-linear gradient (2-80% acetonitrile/0.1% formic acid, flow rate 300 nl/min) on a 50 cm C18 analytical column (75µm i.d., PepMap RSLC, Thermo Scientific). Full MS scans were acquired with resolution 120k and 60k (S. aureus samples) at m/z 400 in the Orbitrap analyzer (m/z range 400-1600 for PAMP experiments; 350-1570 for S. aureus experiments and patient-derived samples) using an AGC target of 500.000 or 50 ms. MS1 parent ions were fragmented by higher energy collisional dissociation (HCD, 30% collision energy) and fragment ion spectra were recorded in the ion trap in rapid mode (AGC=10.000 or 35 ms; for patient-derived samples: AGC=50% or 250 ms). The following conditions were used: spray voltage of 2.0 kV, heated capillary temperature of 275°C, S-lens RF level of 60%. Samples were run in technical triplicates, patient-derived samples in duplicates.

Samples were reconstituted in 0.1% formic acid and peptide concentrations were measured using a NanoDrop spectrometer. 2 µg of tryptic peptides and 0.5 µg of deglycosylated peptides were analyzed in each LC-MS/MS run in duplicates on an Orbitrap Fusion (Thermo Scientific) coupled to a Dionex Ultimate 3000 (Thermo Scientific) nanoelectrospray ion source. Samples were loaded on a 2 cm C18 trap column (Acclaim PepMap100, Thermo Scientific) and separated using a 2.5 h non-linear gradient (2–80% acetonitrile/0.1% formic acid, flow rate 300 nl/min) on a 50 cm C18 analytical column (75 µm PepMap RSLC, Thermo Scientific). Full MS scans were acquired with resolution 120.000 at m/z 400 in the Orbitrap analyzer (m/z range 400–1600, quadrupole isolation). MS1 parent ions were fragmented by higher energy collisional dissociation (HCD, 30% collision energy) and fragment ion spectra were acquired (in the order highest charge to least charge and least intense to highest intensity during a max. 4 sec cycle time) in the ion trap in rapid mode (m/z start 110). The following conditions were used: spray voltage of 2.0 kV, heated capillary temperature of 275 °C, S-lens RF level of 60%, ion selection threshold of 50,000 counts for HCD, maximum ion accumulation times of 50 ms (AGC 5 × 10⁵) for full scans and 35 ms (AGC 1 × 10⁴) for HCD.

Samples were analyzed by nano-LC MS/MS by means of a shotgun strategy using a nano-HPLC (EASY-nLC 1000, Thermo Scientific) coupled online to an LTQ Velos mass spectrometer (Thermo Scientific). Tryptic peptides were injected into an Acclaim® PepMap 100 nanotrap column (75 μm x 2 cm, Thermo Scientific) and separated on a 50 μm x 15 cm C18 Easy Spray column (PepMap® RSLC, 2 μM, 100 Å, Thermo Scientific) at a constant flow rate of 250 nL/min at 45°C. Peptide elution was achieved with a 70 min gradient from 0% to 55% mobile phase B (A: 0.1% formic acid; B: 0.1% formic acid in acetonitrile). Online MS analysis was carried out in data-dependent acquisition mode in two steps: acquisition of full MS scans in positive ion mode with an m/z range from 400 to 1200 Da followed by CID fragmentation of the ten most intense ions using a dynamic exclusion list (exclusion duration 45 s). The following parameters were set: spray voltage, 2.3 kV; capillary temperature, 260°C; normalized collision energy, 35; activation Q energy, 0.25; and activation time, 15 ms.

Generated peptides were separated by nano-flow reversed-phase liquid chromatography (EASY nLC 1000, Thermo Scientific; mobile phase A, 0.1% (v/v) formic acid (FA)/5% (v/v) DMSO; mobile phase B, 100% (v/v) ACN/0.1% (v/v) FA/5% (v/v) DMSO) coupled to a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific). The peptides were loaded onto a trap column (5 mm, PepMap RSLC, C18, 300 μm I.D. particle size 3 μm; Thermo Scientific) and separated with a flow rate of 200 nL/min on an analytical C18 capillary column (50 cm, PepMap RSLC, EASY-spray column, C18, 75 μm I.D. particle size 3 μm; Thermo Scientific), with a gradient of 7–30% (v/v) mobile phase B over 30 min and a column temperature of 45 °C. Peptides were directly eluted into the mass spectrometer.
Typical mass spectrometric conditions were: spray voltage of 2.1 kV; capillary temperature of 320 °C. The LTQ-Orbitrap XL was operated in data-dependent mode. Survey full scan MS spectra were acquired in the orbitrap (m/z 350−1500) with a resolution of 70,000 an automatic gain control (AGC) target at 3 × 10⁶. The ten most intense ions were selected for HCD fragmentation in the orbitrap at an AGC target of 50,000. Singly charged ions and ions with unknown charge states were excluded from the analysis. For cross-linking doubly charged ions were included or excluded in the analysis.