Proxeon easy nlc 2 liquid chromatography system

Each sample was reconstituted in 0.1% FA, sonicated, and 1–2 µg was transferred to MS vials and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using a Proxeon EASY-nLC II liquid chromatography system (Thermo Fisher Scientific, San Jose, CA, USA) coupled with an LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Peptide separation (1 µg) was performed on a reverse phase (RP) column (EASY-Spray column, 50 cm 75 µm ID, PepMap C18, 2 µm particles, 100 Å pore size, Thermo Fisher Scientific) with a 10 mm pre-column (Accucore XL C18, Thermo Fisher Scientific) using 0.1% FA (mobile phase A) and 98% acetonitrile (98% ACN) with 0.1% FA (mobile phase B). A 120 min linear gradient from 5 to 35% B, at a flow rate of 300 nL min⁻¹ was used. A spray voltage of 1.95 kV and a capillary temperature of 230 °C were used for ionization. The peptides were analyzed in positive mode (1 µscan; 400–1600 amu), followed by 10 data-dependent higher energy collision-induced dissociation (HCD) MS/MS scans (1 µscans), using a normalized collision energy of 35% and an isolation width of 3 amu. Dynamic exclusion for 30 s after the second fragmentation event was applied and unassigned charged ions were excluded from the analysis. A total of five samples (n = 5) were independently analyzed.

Peptide fractions were acidified with 0.1% formic acid, cleaned on a C₁₈ MicroSpin column (The Nest Group), and analyzed by LC–MS/MS using a Proxeon EASY-nLC II liquid chromatography system (Thermo Fisher Scientific) coupled to an LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Peptide separation (1 μg) was done as described by Stryiński et al. (37 (link), 39 ).
All acquired MS/MS spectra were analyzed using SEQUEST-HT (Proteome Discoverer 2.4 package; Thermo Fisher Scientific) against a custom-made database containing protein entries for A. simplex plus “Nematoda,” available in the UniProt/TrEMBL database (downloaded November 2019; 1,847,926 entries). The following restrictions were used: full tryptic cleavage with up to two missed cleavage sites and tolerances of 10 ppm for parent ions and 0.06 Da for MS/MS fragment ions. TMT labeling (+229.163 Da on N termini and lysine residues) and carbamidomethylation of cysteine (+57.021 Da) were set as fixed modifications. The permissible variable modifications were methionine oxidation (+15.994 Da), acetylation (+42.011 Da) of the N terminus of the protein, and deamidation (+0.984 Da) of asparagine and glutamine. Moreover, searching parameters included four maximal dynamic modification sites (37 (link)).