Glu fibrinopeptide b

For the liquid chromatography–mass spectrometry analysis (LC–MS), peptides from the digested samples were injected onto a trap column (C18 trap column symmetry 180 μm × 20 mm, Waters, Milford, MA, United States) and were separated in the analytical column (C18 BEH 75 μm × 200 mm, 1.7 mm, Waters, United States) using a capillary UPLC system (nanoAcquity, Waters, United States). The elution gradient of 7–35% phase B (phase A: 0.1% formic acid in water, phase B: 0.1% formic acid in acetonitrile) was performed in 45 min at 275 nL/min. Multiple charged protonated peptides were generated by electrospray ionization and analyzed in a quadrupole time-of-flight mass spectrometer (Synapt HDMS G2, Waters, United States). Data were acquired in the MSE mode, switching from low (4 eV) to high (ramped from 19 to 45 eV) collision energy with scan times of 1.25 s. An external calibration was performed every 60 s using Glu-fibrinopeptide B (Waters, United States).

The protein composition of HDL samples was identified and quantified by the use of a Waters Xevo G2 mass spectrometer (Waters Corporation; MA, USA). In brief, the peptide samples were chromatographically separated on an M-class ultra-performance liquid chromatography (UPLC; Waters Corporation; MA, USA) equipped with an ACQUITY UPLC BEH C18 Column (130 Å, 1.7 µm, 2.1 mm × 50 mm) under gradient conditions at a flow rate of 300 nL/min over 60 min at 35 °C. The mobile phase was composed of acetonitrile as the organic modifier and formic acid (0.1% v/v) for molecule protonation. Peptide fragmentation was performed on a high-definition mass spectrometer (HDMS) instrument equipped with a nano-electrospray ionization (nano-ESI) and operated in the MS^E mode. Parallel ion fragmentation was programmed to switch between low (4 eV) and high (15–45 eV) energies in the collision cell and data was collected from 300 to 3500 m/z utilizing glu-fibrinopeptide B as the separate data channel lock mass calibrant (m/z = 785.8426 Da; Waters Corporation; MA, USA) [39 (link)]. Data were processed with ProteinLynx GlobalServer v3.0 (Waters Corporation; MA, USA) [40 (link)] and Progenesis QI for proteomics (Nonlinear Dynamics, Waters Corporation; MA, USA). Deisotoped results were searched for protein association from the Uniprot (www.uniprot.org) human protein database [41 (link),42 (link)].