Agilent 1100 binary pump

The water: ACN (1:1, v/v) carrier solution was pumped through the system using an Agilent 1100 binary pump (G1312A, Agilent Technologies, Palo Alto, CA, USA) at a flow rate of 0.4 mL min⁻¹. Samples and standards were injected with an Agilent autosampler (G1379A); the injection volume was 20 μL. Analytes were detected by multiple reaction monitoring (MRM) using a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 4000, from Applied Biosystems, AB Sciex, Framingham, MA, USA). Electrospray (ESI) was the ionization source, working in positive mode. Nitrogen was used as a nebulizer and auxiliary gas as well as the collision gas. The source conditions are as follows: Source voltage, 4500 V; source temperature, 500 °C; gas 1 pressure (heating gas at the source), 50 psi; auxiliary gas pressure (drying gas), 50 psi; capillary temperature, 350 °C; curtain gas pressure, 10 psi. Transitions selected and other MS conditions have been detailed in Table S3 (Supplementary Materials). Analyst 6.2 software (AB Sciex) was used to control the instrument and quantify the analytes.
BA standards for calibration were injected at the beginning and end of the FIA-MS sequence. Samples were subsequently injected randomly. Three independent replicates of each sample were analyzed.

HPLC analysis was performed using an Agilent 1100 binary pump (Agilent Technologies Inc., Wilmington, DE, USA), a 1100 micro vacuum degasser, a HP 1050 Autosampler, a HP 1050 variable wavelength detector (operated at 235 nm). The chromatographic separations were achieved on a Supelco SupelcosilTM LC-SI analytical column (4.6 mm × 250 mm, 5 μm) (Supelco Inc., Bellefonte, PA, USA) by using isocratic elution of 25 mM of phosphate buffer with 0.3% of triethylamine at pH 4.7 and ACN with 0.1% of formic acid (30:70, v/v). Effluent was monitored at a wavelength of 254.4 nm, with a flow rate of 1.5 mL/min; the injection volume was 5 µL; retention time of 3.7 min. The column was maintained at 40 °C throughout the analysis.
Standard calibration curves were prepared at different dilutions of CZP: in Milli-Q^® water/ACN (1:1, v/v) with a linear regression coefficient determined in the range 0.5–200 μg/mL, which was 0.9999; in DMSO with a linear regression coefficient determined in the range 0.1–200 μg/mL, which was 1.

HPLC analysis was performed using an Agilent 1100 binary pump (Agilent Technologies Inc., Wilmington, DE, USA), a 1100 micro vacuum degasser, a HP 1050 Autosampler, and a HP 1050 variable wavelength detector (operated at 235 nm). The chromatographic separation was achieved on a Supelco Supelcosil^TM LC-SI analytical column (4.6 mm × 250 mm, 5 μm) (Supelco Inc., Bellefonte, PA, USA) by an isocratic elution of a formic acid 0.1% (v/v) solution in Milli-Q^® water and a formic acid methanol solution (0.1%, v/v) (40:60, v/v). Effluent was monitored at a wavelength of 278.4 nm, with a flow rate of 1 mL/min; the injection volume was 5 µL, retention time of MEL was 3.5 min. The column was maintained at 45.0 ± 0.2 °C throughout the whole analysis.
The standard calibration curves were prepared at different dilutions of MEL in Milli-Q^® water/methanol (1:1, v/v). The linear regression coefficient determined in the range 0.5–200 μg/mL was 0.9999.

The lyophilized peptides were reconstituted in bRPLC solvent A (10 mM triethylammonium bicarbonate, pH 8.5) and fractionated on Xbridge C18 5 um 250 × 4.6 mm column (Waters Corporation, Milford, MA, USA) using Agilent 1100 binary pump (Agilent Technologies, Santa Clara, CA, USA). The sample was resolved through reverse-phase liquid chromatography method using a gradient of 5 to 60% solvent B (10 mM triethylammonium bicarbonate, pH 8.5 in 95% Acetonitrile) with 1 ml flow rate per minute for over 60 min. A total of 96 fractions were concatenated into 12 fractions and vacuum dried. One tenth of each fraction was taken out for total proteomic analysis and the rest was further processed for TiO₂ based enrichment of phosphopeptides. Titansphere beads (GL Sciences, Japan) were mixed with peptides in 1:1 ratio and incubated on rotor for 1 h at room temperature. Peptides were then washed with 80% ACN in 3% TFA and eluted using 4% ammonia solution followed by neutralization with 3% TFA. Eluted peptides were then dried and desalted using C₁₈ stage tips and then subjected to LC-MS/MS analysis.

HPLC analysis was performed using an Agilent 1100 binary pump (Agilent Technologies Inc., Wilmington, DE, USA), a 1100 micro vacuum degasser, a HP 1050 Autosampler, and a HP 1050 variable wavelength detector (operated at 235 nm). The chromatographic separations were achieved on a ZORBAX^® Eclipse XDB-C18 (2.1 mm × 100 mm, 1.8 μm) (Agilent, USA) by using isocratic elution of Milli-Q^® water and acetonitrile (75:25, v/v). Effluent was monitored at a wavelength of 310 nm, with a flow rate of 0.3 mL/min; the injection volume was 5 µL; retention time of RSV was 5.2 min. The column was maintained at 45 °C throughout the analysis.
RSV standard calibration curves were prepared in Milli-Q^® water/EtOH (20% v/v) with a linear regression coefficient determined in the range 0.1–100 μg/mL was 0.9989. All procedures were carried out to protect the sample from light.