C18 material

Analysis of peptide mixtures was performed using a QExactive HF Mass Spectrometer (Thermo-Fisher Scientific). Aliquots containing a 5 µg of total peptides were chromatographed on a 50 cm column with 75 µm inner diameter packed C18 material (Dr. Maisch GmbH, Ammerbuch, Germany). Peptide separation was carried out at 300 nL/min for 90 min using two-step acetonitrile (ACN) gradient of 5–60% over the first 75 min and 60–95% for the following 15 min. The temperature of the column oven was 60 °C. The mass spectrometer operated in data-dependent mode with survey scans acquired at a resolution of 50,000 at m/z 400 (transient time 256 ms). Up to the top 15, 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 value for MS1 and MS2 scan modes was set to 3 × 106 and 105, respectively. The dynamic exclusion was 25 s and 10 ppm. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [93 (link)] with the dataset identifier:PXD011683.

EV samples (100 µL each) were transported to the Proteomic Core Facility, University of Tartu, Estonia in dry ice for proteomic analysis. An amount of 1 µg of protein was injected onto an Easy-nLC 1000 system (Thermo Scientific, Scotland, UK). The sample was eluted at 250 nL/min from the trap to a 75 µm ID × 50 cm emitter-column (New Objective, Littleton, CO, USA) packed with C18 material (3 µm, 300 Å particles, Dr Maisch, Ammerbuch, Germany). The separating gradient was 2–35% B for 60 min and 40–100% B for 5 min (A: 0.1% formic acid (FA), B: 80% Acetonitrile (CAN) + 0.1% FA). Eluted peptides were sprayed onto a Q Exactive Plus (Thermo Fisher Scientific, Waltham, MA, USA) quadrupole-orbitrap mass spectrometer (MS) using nano-electrospray ionization at 2.4 kV (applied through liquid-junction). The MS was operated with a top-5 data-dependent acquisition strategy. Briefly, one 350–1400 m/z MS scan at a resolution setting of R = 70,000 at 200 m/z was followed by five higher-energy collisional dissociation fragmentations (normalized collision energy of 26) of the 5 most intense ions (z: +2 to +6) at R = 17,500. MS and MS/MS ion target values were 3 × 10⁶ and 5 × 10⁴, respectively, with a 50 ms injection time. Dynamic exclusion was limited to 40 s.

Analysis of the peptide mixtures was performed in Orbitrap instrument (Thermo Fisher Scientific, Waltham, Massachusetts, USA) as described previously (Wiśniewski & Mann, 2012). Aliquots containing 5 μg of total peptides were chromatographed on a 50 cm column with 75 µm inner diameter packed C₁₈ material (Dr. Maisch GmbH, Ammerbuch‐Entringen, Germany). Peptide separation was carried out at 300 nl/min for 75 min using a two‐step acetonitrile gradient, 5%–40% over the first 60 min and 40%–95% for the following 15 min. The temperature of the column oven was 55°C.
The mass spectrometer operated in data‐dependent mode with survey scans acquired at a resolution of 50,000 at m/z 400 (transient time 256 ms). Up to the top 15 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 value for MS1 and MS2 scan modes was set to 3 × 10⁶ and 10⁵, respectively. The dynamic exclusion was 25 s and 10 ppm.
The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) with the dataset identifier: PXD006815.

Analysis of peptide mixtures was performed using a QExactive HF mass spectrometer (Thermo-Fisher Scientific, Palo Alto). Aliquots containing 2.5 μg total peptide were chromatographed on a 50 cm column with 75 µm inner diameter packed C₁₈ material (100 Å pore size; Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Peptide separation was carried out at 300 nL/min for 75 min using a two-step acetonitrile gradient 5-40% over the first 85 min and 40-95% for the following 15 min. The temperature of the column oven was 55°C.
The mass spectrometer operated in data-dependent mode with survey scans acquired at a resolution of 50 000 at m/z 400 (transient time 256 ms). Up to the top 15 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 value for MS1 and MS2 scan modes was set to 3×10⁶ and 10⁵, respectively. The dynamic exclusion was 25 s and 10 ppm. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [14 (link)] with the dataset identifier: PXD005230 (for the review: username: reviewer16980@ebi.ac.uk and password: FpqJ9qms).

Peptides from the various fractions were analyzed by a nano-Easy LC (Thermo Fisher Scientific) coupled with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). All peptide fractions were re-suspended in 0.1% formic acid (FA) and loaded onto a 2 cm 100 µm inner diameter pre-column using the nano-Easy LC. Peptides were eluted directly onto the analytical column using a gradient of 0–34% buffer B (90% Acetonitrile, 0.1% FA) over 30–90 min depending on the UV intensity of the individual HILIC fractions. All LC–MS/MS runs were performed using an analytical column of 20 cm × 75 µm inner diameter fused silica, packed with C18 material (Dr. Maisch, Ammerbuch-Entringen, Germany). Mass spectrometry was performed using higher energy collision fragmentation (HCD) fragmentation on a Q-Exactive instrument. MS settings: a full MS scan in the mass area of 400–1,800 Da was performed in the Orbitrap with a resolution of 70,000 FWHM and a target value of 1 × 10⁶ ions. For each full scan the 12 most intense ions (charge states 2–5) were selected for HCD fragmentation and the fragments were detected at a resolution of 17,500 FWHM. Threshold for ion selection was 1.0e4, the AGC target value 2.0e4, activation time was 0.1 ms, isolation window was 1.5 Da, and normalized collision energy was 29.