Orbitrap elite hrms

Digests were analyzed by an Orbitrap Elite HRMS coupled to a Dionex Ultimate 3000 nanoflow LC system via a Flex Ion nanoelectrospray ionization source (Thermo Fisher Scientific, Waltham, MA), as described previously.[5 (link)] A Dionex PepSwitft monolithic column (100 μm i.d. × 25 cm) (Thermo Scientific, Sunnyvale, CA) was used with mobile phases consisting of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), with gradient elution (2–35 % B, 26 min) at a flow rate of 750 nL/min. Two 1- μL aliquots were injected for each sample. Full scan mass spectra were acquired in the positive ion mode with a resolution of 120,000 at m/z 400 in the m/z = 350 – 1500 mass range using the Orbitrap. The MS was operated in data-dependent mode to collect tandem MS (MS2) spectra in the linear ion trap. Additional details of nLC-HRMS analysis are available in the Electronic Supplementary Material (ESM).

For each subject, one 4.7-mm NBS punch was analyzed to detect and quantify HSA-Cys34 adducts as described previously.[11 (link)] Briefly, proteins were extracted from NBS and concentrations of hemoglobin (Hb)[11 (link)] were measured to normalize for blood volume using UV-Visible absorption spectroscopy. Hb concentrations were similar between cases and controls (Table 1), indicating that the NBS punches had comparable blood volumes. Methanol was added to enrich HSA by precipitating Hb and other interfering proteins and the supernatant was digested with trypsin and pressure cycling. The digests were analyzed with an Orbitrap Elite HRMS coupled to a Dionex Ultimate 3000 nanoflow LC system with a nano-electrospray ionization source (Thermo Fisher Scientific, Waltham, MA). The MS was operated in data-dependent mode, and tandem MS (MS2) fragmentation spectra were acquired in the linear ion trap. Samples were analyzed in four batches of ~200 samples, and duplicate injections were made for each sample.