Ultimate 3000 micro hplc apparatus

All the chemicals and reagents used for high-performance liquid chromatography separation coupled to electrospray-ion trap mass spectrometry (HPLC-ESI-IT-MS) analysis were purchased from Sigma-Aldrich (St. Louis, MO, United States). HPLC/low-resolution ESI-MS analyses were performed with a Surveyor HPLC system connected to an LCQ Advantage mass spectrometer (Thermo Fisher Scientific, San Jose, CA, United States). The chromatographic column was a Vydac C8 reverse-phase column (Grace, Hesperia, CA, United States) (2.1 × 150 mm, particle diameter 5 μm). HPLC/high-resolution ESI-MS and MS/MS experiments were carried out using an UltiMate 3000 micro HPLC apparatus (Dionex, Sunnyvale, CA, United States) equipped with an FLM-3000 flow manager module and coupled to an LTQ-Orbitrap Elite or LTQ Orbitrap XL apparatus (Thermo Fisher). The column was a Zorbax 300SB-C8 (1.0 × 150 mm; 3.5 μm particle diameter). All the chemicals and reagents for immunodetection were purchased from Bio-Rad (Hercules, California, United States); the primary mouse monoclonal antibodies (Abs) for α-defensins, cystatin A, cystatin B, S100A8, S100A9, cystatin SN, and thymosin β4 (Tβ4) were purchased from Santa Cruz Biotechnology (Dallas, TX, United States). The secondary rabbit anti-mouse Ab was from Invitrogen (Waltham, MA, United States). Standard Tβ4 was provided by Bachem (Bubendorf, Switzerland).

High-resolution HPLC-ESI-MS/MS experiments were carried out using an Ultimate 3000 Micro HPLC apparatus (Dionex, Sunnyvale, CA, USA) equipped with a FLM-3000-Flow manager module and coupled to an LTQ Orbitrap XL apparatus (Thermo Fisher Scientific, Waltham, MA). The chromatographic column was a Zorbax 300 SB-C8 (3.5 µm particle diameter; column dimension 1 mm i.d. × 15 cm). High-resolution HPLC-ESI-MS experiments were performed by using the following eluents: (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile-water 80/20 (v/v). The applied gradient was 0–4 min 5% B, 4–34 min from 5 to 50% B (linear), 34–54 min from 50 to 90% B (linear), at a flow rate of 80 µL/min. High-resolution positive MS/MS spectra were collected in data-dependent acquisition mode; the three most intense multiply charged ions were selected in a time window of 3 milliseconds and fragmented by using collision induced dissociation (35% normalized collision energy) and spectra were recorded. Tuning parameters were: capillary temperature 250°C, source voltage 4 kV, capillary voltage 48 V, tube lens voltage 170 V.