Zorbax 300sb

AS-48 was purified from cultures of the probiotic strain E. faecalis UGRA10 (Cebrián et al., 2012 (link)) grown on Esprion 300 plus glucose (1%) (DMV Int., Veghel, Netherlands) at maintained pH at 6.55, according to Ananou et al. (2008 (link)). The bacteriocin was purified to homogeneity by RP-HPLC in the conditions previously established (Cebrián et al., 2015 (link)).
Nisin was purified from Nisaplin (Sigma). Briefly, the commercial preparation containing 2.5% nisin was resuspended in H₂O plus 0.05% acetic acid and stirred for 30 min. After this, 0.8 volumes of dichloromethane were added and centrifuged at 650 g for 15 min. The pellet at the interface was recovered and dried. It was resuspended in 0.05% acetic acid, filtered and purified using a preparative chromatographer model Infinity 1260 (Agilent Technologies) and a column Zorbax 300SB-C18 7 μm 21.2 × 250 mm. The sample was applied when the column was equilibrated with 20% solvent B (acetonitrile 0.1% TFA) and separated using a gradient of 25 min from 20 to 65% solvent B at a constant flow rate of 10 mL/min. Solvent A was 0.1% TFA in HPLC-grade water.

Planktonic (N_(L)CSP = 3, single replicate injection) and biofilm (N_(L)CSP = 2, triple replicate injections) peptide resuspensions were separated through HPLC (1260 Infinity LC System, Agilent, Santa Clara, CA) with a C18 column (3.5 μm particle size, 150 mm length × 75 μm internal diameter, Zorbax 300SB, Agilent) and a 60-min increasing organic gradient from 5% to 85% (mobile phase A: 0.1% formic acid in water and mobile phase B: 0.1% formic acid in acetonitrile) for a total run time of 75 min at a flow rate of 250 nL/min. Following chromatographic separation, peptides were simultaneously ionized in the electrospray ionization source at 1.90 kV spray voltage and 275 °C capillary temperature. Peptide ions were analyzed in a high mass resolution, tandem mass spectrometer (Orbitrap Velos Pro, Thermo Fisher Scientific, Waltham, MA) with automatic gain control at 10⁶ ions and between 1 and 200 ms injection time. Full-scan mass spectra were collected from m/z 400–2000 in a data-dependent acquisition mode at 30,000 mass resolution. From each full mass spectrum, ten precursor ions were selected for MS/MS analyses in high energy collision-induced dissociation mode using 30% energy for fragmentation.

For peptic peptide mapping, recombinant OspC_A was diluted with quench solution (200 mM glycine, pH 2.5), and 50 pmol of sample was injected in each run. OspC_A was digested by an in-house-prepared immobilized pepsin column (2.1 by 50 mm) (91 (link)). Digested peptides were trapped and desalted by C₈ (Zorbax 300SB C₈, 2.1 by 12.5 mm, 5-μm particles) for 120 s and separated by a C₁₈ column (Zorbax 300SB 2.1 by 50 mm, 3.5 μm particle diameter, Agilent, Santa Clara, CA). For LC, mobile phase A was 0.1% formic acid in water, and B was 0.1% formic acid in acetonitrile. A total of a 25-min LC method was used: 10 min with 15% to 35% B was used to separate peptides, and 15 min was used for cleaning purposes. Peptides were detected, and the mass was measured with a quadruple time of flight (Q-TOF) mass spectrometer (Agilent 6530 in electrospray ionization (ESI)-positive ion mode). All the peptic peptides were assigned by tandem mass spectrometry (collision induced dissociation [CID] fragmentation). Agilent MassHunter Qualitative Analysis with BioConfirm (version B.07.00) software was used for the analysis of all the mass spectrometry data. A total of 87 peptides were identified and mapped as shown in Fig. S1. This map shows 100% OspC_A sequence coverage with a median length of 17.0 residues and 8.6 average redundancy.

HPLC–DAD analysis was carried out using an Agilent 1260 Series LC system (Palo Alto, CA, USA) equipped with a quaternary pump, a degasser, an autosampler, and a DAD. An XBridge™ BEH C18 analytical column was used (2.5 μm 2.1 × 50 mm; Waters, Milford, MA, USA). Mobile phase A consisted of 0.25% aqueous formic acid, and mobile phase B was ACN. The spectrum of each UV filter is shown in Figure S1. The detection wavelength for OMC, OCT, and EHT was ~300 nm, while the corresponding value for AV and BEMT was ~350 nm, with a flow rate of 0.35 mL/min. The following gradient program was used: 0–1 min, 45–55% B; 1–10 min, 55–70% B; 10–13 min, 70–100% B; 13–25 min, 100% B. The injection volume was 2 μL.
Four kinds of C18 end-capped column were used to determine the chromatographic conditions for the UV filters, including of Chromolith^® Performance (2 × 100 mm; Merck, Darmstadt, Germany), ZORBAX 300SB (3.5 μm 2.1 × 100 mm; Agilent, Santa Clara, CA, USA), XBridge BEH (2.5 μm 2.1 × 50 mm; Waters, USA), and CORTECS (2.7 μm 2.1 × 50 mm; Waters, USA).

The aliquots were solubilized in 15 μL10 mM ammonium acetate, pH 8.0 (buffer A) and fractionated on an HPLC System (Ultimate 3000, Thermo Scientific). Peptides were separated on a 150 mm × 1.0 mm i.d. C18 column (ZORBAX 300SB, Agilent Technologies, Germany) with a 55 min gradient ranging from 2.5 to 42% ACN in 10 mM ammonium acetate, pH 8.0, at a flow rate of 12.5 μL/min. In total, 20 fractions were collected in 60 s intervals in concatenation mode. Each fraction was dried under vacuum and resuspended in 15 μL of 0.10% (v/v) aqueous TFA for nano-HPLC–MS/MS analysis.

INS concentration was analyzed using high-performance liquid chromatography (HPLC) as previously reported (Shrestha et al., 2016 ). Fifty microliters of each sample were injected into an HPLC system (e2695; Waters, Milford, MA, USA) consisting of a pump (W2690/5; Waters), an ultraviolet (UV) detector (W2489; Waters), and a data station (Empower 3; Waters). A C18 column was used (ZORBAX 300SB; 150 × 4.6 mm, 5 μm; Agilent Technologies, Santa Clara, CA, USA), and the mobile system consisted of A [0.1% trifluoroacetic acid (TFA) in distilled water] and B (acetonitrile), with a varied gradient according to the following program: 0 min (80% A), 5 min, (50% A), 7 min (50% A), and 10 min (80% A). Analyses were performed at a flow rate of 1.2 mL/min at 35 °C, and the column eluent was monitored at 214 nm.