Q exactive quadrupole orbitrap

A Q-Exactive™ Quadrupole-Orbitrap™ mass spectrometer (Thermo Fisher Scientific Inc., Rockford, IL, USA) was used to perform PSI experiments. Methanol was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. NNFE was dissolved in methanol at a concentration of 50 μg/mL to prepare a stock solution. Then, the stock solution was diluted to a concentration of 10 μg/mL . Two microliters of the prepared solution was loaded onto a paper tip to perform the PSI experiment. (+) Mode PSI was used over a range of m/z 100–2000. Normal chromatographic paper was used in the PSI experiment. A disposable glass Pasteur pipette (Volac, Poulten & Graf Ltd, UK) was used to load the extracted sample directly onto the centre of the chromatographic paper tip. To make a sharp tip, the chromatographic paper was cut at dimensions of 6-mm base and 14-mm height. A syringe pump (Fusion 100 T, Chemyx, Stafford, TX, USA) was used to load the methanol solvent onto the sample-loaded paper. The flow rate was 40 μL/min, and the voltage directly applied on the paper for ionisation was 3–7 kV. Other parameters for PSI were as follows: spray voltage, 4.5 Kv; capillary temperature, 300 °C; mass resolution 140,000 (FWHM); maximum injection (IT) time, 150 ms; and automatic gain control (AGC), 1 E⁶.

Mass spectrometry analysis of each sample’s peptides was performed using the Q-Exactive quadrupole-Orbitrap (Thermo Fisher Scientific, Waltham, Massachusetts, United States), which was coupled with a Dionex Ultimate 3000 nano-UPLC system as described before by Rossouw.^{12 (link)} Using Xcalibur^TM (version 4.2) (Thermo Fisher, Waltham, Massachusetts, United States), spectral data was collected in a data-dependent manner and details are shown in Supplementary document – Table S1.

For this study, we used 2 mass spectrometers. The Q Exactive Quadrupole Orbitrap (Thermo Fisher Scientific) was used for absolute quantification of APOA1 and APOA4 pool sizes as well as the relative quantification of the HDL proteome across the 6 HDL sizes. We utilized the Orbitrap Fusion Lumos (Thermo Fisher Scientific) for tracer enrichment quantification of APOA4 and APOA1 across 6 HDL sizes. Both instruments were coupled to an EASY-nLC 1000 HPLC pump (Thermo Fisher Scientific). The Q Exactive was fronted with a Nanospray FLEX ion source and the Lumos with an EASY-Spray ion source (Thermo Fisher Scientific).