Superdex peptide 10 300 gl column

The degradation products of the FBS of strain A3 towards L. japonica piece and L. japonica roots were identified by high-resolution LTQ-Orbitrap-MS (Thermo, USA) in the negative-ion mode. The source voltage and capillary temperature were set at 3.6 kV and 275 °C, respectively. The mass acquisition range was 160–1500 m/z or 200–1500 m/z.
The AOs composition in the L. japonica root hydrolysate produced by the FBS of strain A3 was analyzed by HPLC on a Superdex Peptide 10/300 GL column (GE Healthcare, USA) [45 (link)]. The AOs in the hydrolysate were eluted with 0.2 M NH₄HCO₃ at a flow rate of 0.3 ml/min and detected by a UV detector at 210 nm. The commercial saturated mannoronate oligomers were taken as the standards. The proportion of each AO was calculated according to the percentage of the AO area to the total chromatograph area of the AOs [46 (link)].
Total sugar content of the degradation products was measured by the phenol–sulfuric acid method [47 (link)] using d-glucuronic acid as the standard. The content of uronic acid in the degradation products was analyzed by the sulfamate/m-hydroxydiphenyl assay described by Filisetti-Cozzi [37 (link)] using d-glucuronic acid as the standard.

The cAbVCAM1-5 Nb was modified by incubation with a 12-fold molar excess of TFP-RESCA (135 mM stock in DMSO) in 0.05 M sodium carbonate buffer (120 μM of Nb, pH 8.5–8.7) for 2 h at RT. The modified Nb was subsequently purified by SEC on a Superdex Peptide 10/300 GL column (GE Healthcare, Belgium) and eluted in 0.1 M NH₄OAc pH 7 on a medium-pressure chromatography system (Bio-Rad NGC, Belgium). The average number of chelators per Nb was determined by ESI-Q-ToF-MS (GIGA Proteomics, Liège, Belgium).

The nitrogen content was analyzed by the Kjeldahl method (ISO 5983-2, 2009 [49 ]) and the crude protein content was estimated on the basis of N × 6.25. The ash was determined by combustion of the raw material at 550 °C (ISO 5984-2, 2002 [50 ]). The dry matter content was determined by drying at 103 °C (ISO 6469-2, 2002 [51 ]). The fat content of the protein hydrolysate was analyzed by the EU method (Commission Directive 98/64/EC), while the fat content of the raw material was analyzed based on ethyl acetate extraction (NS 9402). Peptide size distributions were measured by HPLC size exclusion chromatography (SEC) (1260 series HPLC Agilent Technologies, Santa Clara, CA, USA) using a Superdex Peptide 10/300GL column (GE Healthcare, Uppsala, Sweden). The eluent was acetonitrile with TFA and UV detection at 190–600 nm [52 (link)].

Size-exclusion experiments were performed at room temperature using a Superdex Peptide 10/300 GL column (GE Healthcare Life Sciences) by injecting 100 μl of a 50 μM (total peptide concentration) sample in 10 mM potassium phosphate and 100 mM potassium fluoride, pH 7, at a flow rate of 0.5 ml/min. Elution profiles were recorded via A₂₈₀.

For LC-MS analysis, the treated/ctrl peptide libraries were purified by SEC. To this end, the sample was loaded onto a pre-equilibrated Superdex Peptide 10/300 GL column (GE Healthcare) with a flow rate of 0.5 mL/min, installed in an ÄKTA explorer system coupled to an automated fractionation system. A 0.1 M ammonium acetate buffer (pH 5) was used throughout the column equilibration and purification and the elution profile was recorded at 280 nm. The fractions of the library peak were collected, pooled together and concentrated by vacuum centrifugation overnight. The dried peptide libraries were then further analyzed by LC-MS/MS measurements.

The peptide apparent molecular weight (MW) distributions of oral, gastric and intestinal aliquots were obtained by SEC using a Superdex Peptide 10/300 GL column (GE Healthcare, Uppsala, Sweden) on an AKTA Purifier system (GE Healthcare). SEC was carried out in isocratic conditions with an elution solution of 30% acetonitrile, 69.9% ultrapure water and 0.1% TFA solvent at a flow rate of 0.5 mL.min⁻¹. Oral, gastric and intestinal aliquots were first diluted in ultrapure water (18.5 g L⁻¹, w/v) and subjected to a magnetic stirring for 15 min. The diluted samples were then centrifuged at 15,000× g for 15 min and the supernatants were filtered through a 0.22 µm membrane filter before injection. The absorbance was monitored at 214 nm for 70 min. The column was calibrated with the following standard peptides: cytochrome C (12,327 Da), aprotinin (6511 Da), insulin beta-chain (3496 Da), neurotensin (1673 Da), substance P (1348 Da), substance P fragment 1–7 (900 Da) and leupeptin (463 Da).

Typically, 0.5 mL of a 60 μM Zn₆E_c-1 solution was incubated with proteinase K using a molar ratio of 50:1 in 50 mM Tris-HCl, 10 mM CaCl₂, pH 8.0, at room temperature for 40 min without mixing. The digestion mixture was immediately applied to a Superdex Peptide 10/300 GL column (GE Healthcare) that was pre-equilibrated with 10 mM ammonium acetate, pH 7.5. Fractions containing cleaved protein fragments were collected for further analysis. For the analysis of Cu(I) binding, the corresponding amount of Cu(I) (see below) was added prior to proteolytic cleavage and 1% acetonitrile was added to the column buffer. Protein concentrations were determined by thiol group quantification with the 2-PDS assay assuming only five accessible Cys for the γ-domain after addition of 1 or 2 equiv. of Cu(I) based on control experiments and 10 accessible Cys for the fraction containing the β_E-domain after addition of 2 equiv. of Cu(I) (see also Table S1) [29 (link)]. Metal ion concentrations were determined with F-AAS in 0.2 M HNO₃ using an AA240FS spectrometer (Agilent Technologies AG, Basel, Switzerland).