Acquity classic uplc

All size exclusion chromatography experiments were performed on Waters Acquity Classic UPLC, equipped with a multi-wavelength detector. Protein species separation by an isocratic elution was achieved using a flow rate of 0.4 mL/min on a BEH SEC column (200 Å, 1.7 µm, 4.6 mm × 150 mm, Waters, Baden, Switzerland) maintained at 40 °C. The mobile phase was 50 mM sodium phosphate, 400 mM sodium perchlorate, and pH6.0. The detection of signals was performed at 280 nm.
The obtained chromatograms of individual protein components and complexes in PBS matrix were evaluated directly. For complexes incubated in a matrix containing mouse serum, a subtraction procedure was introduced. Hereby, chromatograms of mouse serum control samples (addition of PBS instead of protein components) were subtracted from the chromatograms of samples containing formed complexes in mouse serum matrix prior to the data interpretation. Data collection, evaluation, integration, and processing by subtraction were performed with Chromeleon 7.3 (Thermo Scientific, Reinach, Switzerland) software.

The quantification of Gly-Sar in cell homogenates was performed on a UPLC-MS/MS system consisting of a triple-stage quadrupole mass spectrometer (Waters Xevo TQ-XS with Z-spray ESI source) and an Acquity Classic UPLC® (Waters, Milford, MA, USA). Chromatographic separation was achieved on a Waters Cortecs UPLC® HILIC column (90 Å, 1.6 µm, 2.1 × 50 mm) heated to 60 °C with the injection volume set to 10 µL and a flow rate of 0.4 mL/min. The mobile phase consisted of H₂O/ACN (95/5, v/v) + 0.1% FA (aqueous eluent; A) and ACN + 0.1% FA (ACN eluent; B). Gly-Sar was eluted with an isocratic composition of 16% A/84% B for 3 min. Subsequently, the conditions were changed to 50% A/50% B within 0.5 min and maintained for 0.5 min to flush the column. Afterwards, the initial ratio of 16% A and 84% B was restored during 0.5 min. The mass spectrometer was operated using an electrospray ionization (ESI) source in the positive ion detection mode. Quantification of Gly-Sar and IS was performed by selected reaction monitoring (SRM) using argon collision gas for collision-induced dissociation (CID). Mass spectrometric parameters were optimized for the detection of Gly-Sar and the IS using the auto-optimization feature IntelliStart of the MassLynx V4.2 system software (Waters, Milford, MA, USA). Optimized mass spectrometric conditions are shown in Table 1.