6495 qqq mass spectrometer

Pancreatic islets were prepared (SI Appendix) and analyzed using an Agilent 1290 series Ultra-high-performance liquid chromatography system connected to an Agilent 6495 QQQ mass spectrometer after separation on a ZORBAX Eclipse plus C18 column (2.1 × 50 mm, 1.8 µm, 95 Å, Agilent) at 40 °C (SI Appendix). Mass spectrometry analysis was performed in positive ion mode with dynamic, scheduled multiple reaction monitoring (MRM). Mass spectrometry settings, LC-MS gradient, and MRM transitions for each lipid class were adapted from a previously published method (63 (link)). Data analysis was performed on Agilent MassHunter Quantitative analysis software. Relative quantitation was based on one-point calibration with Ceramide d18:1/8:0. The data were further normalized to the total number of islets in each sample.

The individual samples used in the quantitative mass spectrometry assay were unique compared to the samples utilized for the discovery section. The demographic details for this cohort are described in Table 3. Heavy isotopically labelled peptides were obtained as part of the BAK76 protein target kit from MRM-Proteomics (BAK76-kit, Canada). Samples were processed using the manufacturer’s protocol (additional details in supplemental materials). Liquid chromatography mass spectrometry was conducted on with a 1290 UHPLC coupled to a 6495 QQQ mass spectrometer (Agilent Technologies).
Statistical analyses were conducted using: Progenesis QI (Waters, UK) for the 2D gel data; GraphPad Prism v 8.0 for Mac OS X (GraphPad Software, USA) for the ROC; t-test analysis: One-way ANOVA with Sidak correction of multiple comparisons and Chi-squared test. A p-value < 0.05 was considered significant.

The analysis of the target compounds
was performed using an Agilent 1260 Infinity II SFC system coupled
with a 6495 QqQ-mass spectrometer using both electrospray ionization
(ESI) and atmospheric pressure chemical ionization (APCI) sources
(Agilent Technologies, Waldbronn, Germany). The UHPSFC-MS system was
controlled with the MassHunter Workstation Software v. 10.0 from Agilent
Technologies. Data analysis was carried out using the same software.
Separation of the vitamins was achieved on a Torus 1-aminoanthracene
(1-AA) column (100 mm × 3.0 mm, 1.7 μm) with a Torus 1-AA
pre-column (5 mm × 2.1 mm, 1.7 μm), both from Waters Chromatography.
Experimental design was performed using the software Umetrics MODDE
v. 12.1.0.3948 from Sartorius Stedim Biotech.