Accela 600 hplc system

The enriched O-glycopeptides from bovine fetuin digests were analyzed on a nano-ESI-Q-TOF mass spectrometer (Waters, Manchester, United Kingdom) with collision-induced dissociation (CID) in a positive mode. Full scan MS data were obtained at m/z 600–1700.
The enriched bovine fetuin and HKU1 S1 O-glycopeptides were separated and characterized using Q-Exactive Orbitrap coupled with Accela 600 HPLC system (Thermo, CA, United States), respectively. For the separation of peptides with reverse-phase liquid chromatography, 0.1% FA (pH 2.59) aqueous solution and ACN/0.1% FA were used as mobile phases A and B, respectively. The analytical column with an inner diameter of 75 μm was packed in-house with C18 AQ particles (3 μm, 120 Å) to 12 cm length. The flow rate was set at 600 nl/min. Gradient elution was performed with 2–8% B in 0.2 min, 8–50% B in 45 min, 50–90% B in 0.5 min, and 90% B in 5 min. Full mass scans were carried out on the Orbitrap with acquisition range from m/z 500 to 1500 (R = 70,000 at m/z 400). The 20 most intense ions from the full scan were selected for fragmentation via higher-energy collisional dissociation (HCD) in the ion trap. The dynamic exclusion function was set as follows: repeat count 1, repeat duration 30 s, and exclusion duration of 60 s.

LC-20AT HPLC system (Shimadzu, Japan) was used for the analysis of anthocyanins according to our published paper [6 (link),31 (link)]. An Agilent SB-C18 column (250 × 4.6 mm², 5 µm, Santa Clara, CA, USA) was used for the separation of anthocyanins with a flow rate of 1 mL/min. The injection volume was 20 µL and the column temperature was maintained at 35°C. The mobile phase consisted of (A) 2% formic acid in water and (B) acetonitrile. The gradient program was set as follows: 0–30 min, 0–20% B; 30–45 min, 20–35% B; 45–46 min, 35–100% B; 46 51 min, isocratic 100% B; 51–52 min, 100% to 0% B; and 52–57 min, isocratic 0% B. Malvidin-3-O-glucoside was used as the external standard for quantitation of individual anthocyanins. An Accela 600 HPLC system coupled with a Thermo Fisher LTQ XL ion trap mass spectrometer (Thermo Fisher Scientific Inc, San Jose, CA, USA) was used for the identification of anthocyanins in the wine samples based on the published methods [14 ,23 (link)].

MSC were seeded on nanotopographies at 1000 cells/cm² and allowed to grow for 11 days. Cells were washed and incubated in basal media comprising 50% normal glucose and 50% ¹³C₆-Glucose (Cambridge Isotopes Ltd) for a further 3 days. Extractions were performed as above. LC-MS was performed as previously described⁶⁹. Briefly, the LC-MS platform consisted of an Accela 600 HPLC system combined with an Exactive (Orbitrap) mass spectrometer (ThermoFisher). Two complementary columns were used; the zwitterionic ZIC-pHILLIC column (150 mm × 4.6 mm; 3.5 μm, Merck) and the reversed phase ACE C18-AR column (150 mm × 4.6 mm; 3.5 μm Hichrom) and in both cases sample volume was 10 μl at a flow rate of 0.3 ml/min. Eluted samples were then analysed by mass spectrometry.
LCMS data of ¹³C-labelled extracts were processed to generate a combined PeakML file as described previously^{65 (link)}. Further analysis using mzMatch-ISO in R⁶⁶ generated a PDF file containing chromatograms used to check peak-shape and retention time. A tab-delineated file detailing peak height for each isopotologue was also generated to calculate percentage labelling.