Agilent 1290 uhplc system

UPLC-MRM/MS analysis was performed using a commercial service facility at Creative Proteomics.10 μL aliquots of the resultant solutions were injected into a C18 LC column (2.1 × 150 mm, 1.8 μm) to run UPLC-MRM/MS on an Agilent 1290 UHPLC system coupled to an Agilent 6495B QQQ mass spectrometer operated in the positive-ion mode, with the use of 0.1% for mica acid in water (A) and acetonitrile (B) for binary gradient elution (50% to 100% B in 15 min), at 0.35 mL/min and 55 °C. The resulting data are normalized to input cells and expressed in per cell basis.

Peptides (approximately 15 µg) were fractionated using an Agilent 1290 UHPLC system (Agilent Technologies, Santa Clara CA) with an in-house packed capillary column. LC mobile phases were: (A) ammonium formate (10 mM, pH 7.9) and (B) water (10% v/v) and acetonitrile (90% v/v). Fractions were monitored by total UV absorbance.

UHPLC-MS analysis was performed on an Agilent 1290 UHPLC system (Agilent Technologies) which was equipped with TripleTOF 6600 mass spectrometry (AB Sciex). Sample was separated on a UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm, Waters) with column temperature at 25 °C. The injection volume was 2 μL for each sample. Mobile phase A was acetonitrile. Mobile phase B consisted of ammonium acetate and ammonia hydroxide in water (25 mmol/L, respectively, pH = 9.75). Gradient elution was applied (0–0.5 min, 95% A; 0.5–7.0 min, 95%-65% A; 7.0–8.0 min, 65%-40% A; 8.0–9.0 min, 40% A; 9.0–9.1 min, 40%-95% A; 9.1–12.0 min, 95% A). The mass spectrometry was in tandem with UHPLC via an electrospray ion (ESI) source to acquire MS and MS/MS spectra under IDA mode. In this mode, the top 12 precursor ions from each MS scan (m/z 60–1200) were chosen for MS/MS scan (m/z 25–1200) at collision energy of 30 eV. The cycle time was 0.56 s. Gas 1, gas 2, and curtain gas of the ESI sourse was 60, 60, and 35 psi, respectively. The source temperature was 600 °C. The ion spray voltage was 5000 V and -4000 V in positive and negative ion modes, respectively.

Experiments were performed on an Agilent 1290 UHPLC system (Agilent Corporation, United States) and an API 3200 triple-quadrupole mass spectrometer (Concord, Ontario, Canada). A ZORBAX Eclipse XDB-C18 (2.1 mm × 100 mm, 1.8-Micron, Agilent) was used for chromatographic separation. The mobile phase consisted of formic acid aqueous solution (0.1%, v/v) (A) and acetonitrile (B), with application of the gradient elution as follows: 0–5 min, 10%–26% (B); 5–7 min, 26%–27% (B); 7–11 min, 27%–35% (B); and 11–13 min, 35%–95% (B). The column temperature and injection volume were set at 35°C and 2 μL, respectively. The flow rate was maintained at 0.3 mL/min. The key parameters of the electric spray ion source (ESI) in positive and negative ion modes were optimized, and the optimal results were as follows: curtain gas (CUR), 15 psi; ion spray voltage (IS), ± 4500 V; ion source temperature (TEM), 550°C; gas1 (GS1), 45 psi; gas2 (GS2), 25 psi. The MS parameters of each compound, including the declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) are shown in the Supplementary Table S1. Chromatograms of the ten compounds and internal standards, in sample solution and working standard solutions, respectively, are shown in Figure 1.

Measurements on the ZIC-pHILIC column were done on an Agilent 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, USA) consisting of a quaternary pump, an autosampler, and a diode array detector with a flow cell of 1 μL. Measurements on the capillary monolithic columns were executed on an Ultimate 3000 RSLC nano system (Dionex, Amsterdam, the Netherlands), with a Binary Rapid Separation Nano Flow pump with nano flow selector, an autosampler, a four-port injection valve with a 20 nL internal loop (VICI, Houston, TX, USA) and a variable wavelength detector (VWD) with a 3 nL flow cell. Experiments were executed at room temperature (21.5 ± 0.5 °C), using an injection volume of 20 nL for the monolithic columns, and 1 μL for the ZIC-pHILIC column. The detection wavelength was set to 254 nm, and the data acquisition rate was 40 Hz for all experiments. Data acquisition and processing were done with Chromeleon software (version 6.8, Dionex) or OpenLab Chemstation software (edition C.01.07, Agilent Technologies).
pH values were measured using a Metrohm 691 pH meter (Antwerp, Belgium). Scanning electron microscopy (SEM) experiments were performed using a TESCAN MIRA4 system (Brno, Czech Republic), using an energy between 5 and 15 keV. Magnifications were between 700× and 50.000×.