Zorbax 300sb c8

HPLC was used to determine relative quantities of gliadins and glutenins in a sample. To obtain a standard curve using HPLC, increasing concentrations of bovine serum albumin (BSA; 0, 10, 25, 50, 75, 100, 150, and 300 mg/mL) were loaded onto the C8 reversed-phase analytical column (Zorbax 300SB-C8, Agilent Technologies) and resolved following Mejías et al. (38 (link)). Two peaks were observed, respectively, at 16.78 and 17.98 min retention times. Peaks were integrated, and the obtained peak areas in milli-absorbance units (mAU) were regressed against the BSA concentrations loaded onto the HPLC column to obtain a standard curve. The gliadin and glutenin extracts of the control and DME TILLING mutants were analyzed analogously as mentioned above for BSA, and the peak areas were calculated. The mAU values were plotted on the standard curve to determine relative protein concentrations, and the values obtained for each peak were summed to calculate a cumulative concentration for each of the glutenin and gliadin fractions from control and mutant lines.

Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC/ESI-MS) was performed on a 6210 LC-TOF spectrometer coupled to a HPLC system (Agilent Technologies). All solvents used were HPLC grade (Chromasolv, Sigma-Aldrich), trifluoroacetic acid (TFA) was from Acros Organics (puriss., p.a.). Just before analysis protein samples were diluted in acidic denaturing conditions to a final concentration of 5 µM with solution A (0.03% TFA in water). Solvent B was 95% acetonitrile-5% water-0.03% TFA. Protein samples were firstly desalted on a reverse phase-C8 cartridge (Zorbax 300SB-C8, 5 μm, 300 µm ID´5 mm, Agilent Technologies) for 3 min at a flow rate of 50 μl/min with 100% solvent A and then eluted and separated onto a RP-C8 column (Jupiter, 5 μm, 300 Å, 1 mm ID × 50 mm, Phenomenex) at a flow rate of 50 μl/min using the following linear gradient: from 5 to 95% solvent B in 15 min, then remaining 2 min at 100% solvent B and finally re-equilibrating the column at 5% solvent B for 10 min. MS acquisition was carried out in the positive ion mode in the 300-3200 m/z range. MS spectra were acquired and the data processed with MassHunter workstation software (v. B.02.00, Agilent Technologies) and with GPMAW software (v. 7.00b2, Lighthouse Data, Denmark).

A method to quantify 329 from plasma has been developed using an internal standard, liquid–liquid extraction, and HPLC-MS/MS. Mouse plasma samples were prepared from treated mice at the indicated times frozen at -80°C until analysis. Plasma samples were thawed (20 µl) and transferred to polypropylene tubes, and the internal standard is added (20 µl of 0.1 ng/µl). Samples were diluted in 0.1 M phosphate buffer (pH = 7.4) and equal volume of methyl tertiary butyl ether. The samples were mixed and centrifuged at 12,000×g for 5 min, and the supernatant was transferred to a clean polypropylene tube. The solvent was evaporated to dryness, brought up in mobile phase analyzed by HPLC-MS/MS (ABSciex 4000). The mobile phase is delivered via gradient using acetonitrile and 0.1% formic acid on an Agilent Zorbax 300SB-C8 150 × 4.6 mm, 5-µm column. The mass spectrometer utilized an electrospray ionization probe run in positive mode. Multiple reaction monitoring was employed with Q1/Q3 (m/z) transitions for 329 at 718.2/128.1 and 687.3/128.1 for the internal standard. The lower limit of quantification is 0.1 ng/ml using 20 µl of plasma.

The peptides and antibody conjugates were analyzed using a single quadrupole mass spectrometer (Agilent: G7129A) coupled with 1260 infinity II Quaternary Pump (Agilent: G7111B). (Column: Agilent ZORBAX 300SB-C8 5 μm 4.6×150 mm; Pursuit 5 Diphenyl 150×2.0 mm) The elution conditions for peptides were as follows: mobile phase A= 0.1% formic acid water; mobile phase B= 0.1% formic acid in acetonitrile; gradient 0-0.1 min, 10-15% B; 0.1-8 min, 15-65% B; 8-8.5 min, 65-10% B; flow rate= 1 mL/min. The elution conditions for antibody conjugates were as follows: mobile phase A= 0.1% formic acid water; mobile phase B= 0.1% formic acid in acetonitrile; gradient 0-0.1 min, 10-15% B; 0.1-8 min, 15-50% B; 8-8.1 min, 50-10% B; flow rate= 0.5 mL/min. The absorbance was measured at 280 nm. Automatic data processing was performed with MassHunter BioConfirm software (Agilent) to analyze the intact and reduced MS spectra.

Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) was performed on a 6210 LC-TOF spectrometer coupled to a HPLC system (Agilent Technologies). All solvents used were HPLC grade (Chromasolv, Sigma-Aldrich), trifluoroacetic acid (TFA) was from Acros Organics (puriss., p.a.). Solvent A was 0.03% TFA in water; solvent B was 95% acetonitrile-5% water-0.03% TFA. Just before analysis, MBP-ZEBRA samples (10 μM in phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) containing 0 or 20 mM DTT were diluted to a final concentration of 5 μM with water and 4 μl were injected for MS analysis. Protein samples were first desalted on a reverse phase-C8 cartridge (Zorbax 300SB-C8, 5 μm, 300 μm ID × 5 mm, Agilent Technologies) for 3 min at a flow rate of 50 μl/min with 100% solvent A and then eluted with 70% solvent B at flow rate of 50 μl/min for MS detection. MS acquisition was carried out in the positive ion mode in the 300–3200 m/z range. MS spectra were acquired and the data processed with MassHunter workstation software (v. B.02.00, Agilent Technologies) and GPMAW software (v. 7.00b2, Lighthouse Data, Denmark).

Purified sHB-EGF (30  μg) was freeze-dried and dissolved in 150 μl of dissolving buffer (water plus 0.1% trifluoroacetic acid). For each injection, 50 μl of the sample was used, and the volume of the sample loop is 20 μl. As for column, Agilent ZORBAX 300SB-C8 was chosen. The mobile phase A was acetonitrile plus 0.1% trifluoroacetic acid, and the phase B was water plus 0.1% trifluoroacetic acid. The elution gradient was set as: 0 min, 10% A plus 90% B; 30 min, 100% A. The flow rate was set at 1 ml/min and the column temperature was set at 30 °C. The detection wavelength was 280 nm. The retention times and the peak areas were analyzed with the software supplied by the manufacturer.

LC-MALDI-TOF-MS experiments were performed on an ultrafleXtreme MALDI-TOF/TOF (Bruker, Bremen, Germany). Peptides were separated via an Agilent 1200 by C8 (Zorbax 300SB-C8, 3.5 μm, 100 × 0.3 mm from Agilent) and C4 (Phenomenex, Jupiter C4 300A, 150 × 0.3 mm) capillary reversed phase separation. Typically, 4–8 μg of samples were injected. Buffer A was 0.1% TFA. Buffer B was ACN with 0.1% TFA. The 40 min gradient was as followed: Buffer B: 5–20% in 5 min and 20–55% over 30 min. Fractions of 3 μL were spotted every 15 seconds by a PROTEINEER fc II on a MTP BigAnchor 384 MALDI-target (both Bruker, Bremen, Germany). Super dihydroxybenzoic acid (sDHB) was used as matrix. Aliquots of 0.5 μL of a 50 mg/mL matrix solution in 50% ACN/water/0.1% TFA were spotted to each dried fraction.
The fractions were then analysed in an automated way by WARP-LC 1.3 and Compass 1.4 in positive linear ion mode and spectra were displayed according to their retention time in the SurveyViewer of the software. In-source decay (ISD) spectra were then manually acquired on selected fractions using a positive reflector method optimised for ISD. Typically, 5000–10000 laser shots were accumulated for each ISD spectrum. External calibration of ISD spectra was performed with ISD fragments of bovine ubiquitin spotted on the calibration chips of the MTP 384 BigAnchor target.