Thermo ltq velos orbitrap

Plasma samples were thawed and analyzed by LC-MS at Emory University as previously described.8 (link)–10 (link, link),15 ,16 (link) Samples were randomized into batches of 20 prior to analysis. Pooled reference plasma was run prior to and after each batch for quality control and assurance. Plasma sample aliquots (65 μL) were treated with 130 μL acetonitrile (2:1 vol/vol) containing 3.5 μL of an internal isotopic standard mix,10 (link),15 ,17 (link) placed on ice for 30 minutes, and centrifuged for 10 minutes (16,100g at 4°C) to remove protein. The supernatants were loaded onto a Shimadzu autosampler maintained at 4°C and analyzed in triplicate using a Thermo LTQ Velos Orbitrap (Thermo Scientific, San Jose, CA, USA) and C18 column chromatography. Elution was obtained with a formic acid/acetonitrile gradient at a flow rate of 0.35 mL/min for the initial 6 minutes and 0.5 mL/min for the remaining 4 minutes. The first 2-minute period consisted of 5% solution A (2% [vol/vol] formic acid in water), 60% water, 35% acetonitrile, and the final 4-minute period was maintained at 5% solution A, 95% acetonitrile. The mass spectrometer was set to collect mass-to-charge ratio (m/z) from 85 to 2000 over 10 minutes at 60,000 mass resolution. Electrospray ionization was used in positive mode for detection.9 (link),10 (link),15 ,17 (link)

The analysis was performed using a Thermo LTQ Velos Orbitrap high resolution mass spectrometer (Thermo Scientific, Pittsburgh, PA, USA) equipped with a heated ESI ion source. The mass scan range was set to 100–2000 m/z, with a resolving power of 100 ​000. The m/z calibration of the LTQ-Orbitrap analyzer was performed in the positive ESI mode using a solution containing caffeine, MRFA (met-arg-phe-ala) peptide and Ultramark 1621 according to the manufacturer’s guidelines. The ESI was performed with a heated ion source equipped with a metal needle and operated at 4 ​kV. The source vaporizer temperature was adjusted to 400 ​°C, the capillary temperature was set at 270 ​°C, and the sheath and auxiliary gases were optimized and set to 40 and 20 arbitrary units, respectively.
The separation of the biological extracts was performed using an Eclipse XDB C18 150 ​× ​4.6 ​mm column packed with 5 ​μm particles size. The separation was achieved using a gradient composed of water/methanol. The mobile phase solvents were composed of A: 100% water ​+ ​(0.1% formic acid) and B: 100% methanol ​+ ​(0.1% formic acid). The gradient elution program is summarized in Table 1. The injection volume was 10 ​μL and the flow rate was set to 400 ​μL/min. XcaliburTM software (Thermo Scientific) was used for method development and data treatment.

Ion dissociation mass spectrometry was performed for representative discriminating metabolites detected in the study samples. Samples were analyzed using a Thermo LTQ Velos Orbitrap high-resolution (60,000 mass resolution) mass spectrometer (Thermo Fisher Scientific, San Diego, CA, USA) operated in positive ion mode with 10-minute C18 column chromatography and standard source conditions used for the untargeted metabolic profiling. Before analysis, plasma proteins were precipitated using acetonitrile (2:1 vol/vol) and allowed to equilibrate for 30 minutes. Collision-induced ion dissociation was accomplished using high purity N₂ at a normalized collision energy of 35%. The tandem mass spectrometry data were processed using the xcmsFragments function in XCMS,^{31 (link)–33 (link, no link found)} and the experimental spectra were compared with in-silico fragmentation using MetFrag.³⁴