Picotip emitter silica tip

The peptides were analyzed using the Nano-LC system Ekspert nLC415 (Eksigent Technologies, Dublin, CA, USA) equipped with AB SCIEX 5600 triple TOF mass spectrometer (AB SCIEX; Concord, Canada). The injection volume was 2 µL and the peptides were trapped on a Nano-LC trap column (0.5 mm × 350 µm; 3 µm; Eksigent Technologies) for 7 min through the mobile phase A, which consisted of 0.1% FA in 100% HPLC water. The separation of peptides was then conducted using an analytical column (150 mm × 75 µm; 3 µm; Eksigent Technologies), which was linked with a nanospray tip (PicoTip Emitter Silica Tip by New Objective, Woburn, MA, USA). The flow rate was set at 300 nL/min for 120 min with mobile phase B, which consisted of 0.1% FA in ACN and set with a linear gradient: 5%–40% for 105 min, 40%–90% for 0.5 min, 90% for 6 min, 90%–5% for 0.5 min, and 5% for 8 min. β galactose (50 mol) was used for autocalibration after every three injections. The Q-TOF conditions used were as follows: ion source gas and curtain gas were set to 12 and 25, respectively, ion spray voltage floating was set to 2200 V, and interface heater temperature was set to 150 °C.

The samples were analyzed using the Nano-LC system Ekspert nLC415 (Eksigent Technologies, Dublin, CA, U.S.A.) coupled to the AB SCIEX 5600 triple TOF mass spectrometer (AB SCIEX, Concord, Canada). The LC solvents used were mobile phase solution A (0.1% formic acid in HPLC-grade water) and mobile phase B (0.1% formic acid in HPLC-grade acetonitrile). Next, 2 μl of samples were injected into the NanoLC trap column (0.5 mm × 350 µm; 3 µm; Eksigent Technologies) and eluted from the analytic column (150 mm × 75 µm; 3 µm; Eksigent Technologies) to the nanospray tip (PicoTip Emitter Silica Tip by New Objective, Woburn, MA, U.S.A.) to ionize. Total gradient time was 120 min, during which the mobile phase solution B was 5–40% for the first 105 min, followed by 40–90% for 0.5 min, 90% for 6 min, 90 to 5% for 0.5 min, and 5% for the last 8 min, at a constant flow rate of 300 nl/min. Further, 50 fM β-galactose was analyzed once for every three samples as auto calibration. The parameters were set to 12 ion source gas, 25 curtain gas (CUR), 2200 V ion spray voltage floating, and 150°C interface heater temperature. Data were analyzed in positive ion mode.

Proteins in ECM hydrogels and in the native umbilical cord tissue were identified by high-resolution LC–tandem mass spectrometry (MS/MS). The isolated proteins were digested with trypsin, concentrated and desalted using a trapping column (100 μm × 30 mm) filled with 3.5-μm X-Bridge BEH 130 C18 sorbent (Waters) and eluted onto an analytical column (Acclaim Pepmap100 C18, 3 µm particles, 75 μm × 500 mm; Thermo Fisher Scientific, Waltham, MA, USA). The analytical column outlet was directly connected to the Digital PicoView 550 ion source with PicoTip emitter SilicaTip (New Objective; FS360-20-15-N-20-C12). The analysis of the mass spectrometric RAW data files was carried out using the Proteome Discoverer software (Thermo Fisher Scientific; version 1.4) (Supplementary Materials).