Starting from 20 μl of serum samples, prepared according to the method of Melo et al. (46 (link), 47 (link)), patients and control groups were evaluated in quintuplicates through direct injection into a high-resolution mass spectrometer (HRMS; ESI-LTQ-XL Orbitrap Discovery instrument [Thermo Scientific, Bremen, Germany]). Instrumentation parameters were set as follows: sample flow of 10 μl/min, sheath gas at 10 arbitrary units, source voltage of 5 kV, and capillary temperature of 280°C. Analytical quintuplicates were prepared and analyzed for each sample, from which metabolic fingerprints were captured in the mass range of 750 to 1,700 m/z in the positive ion mode.
Intending to confirm our findings, tandem mass spectrometry (MS/MS) was applied in the same instrument mentioned above. The collision gas used was helium, with collision-induced dissociation energy ranging from 30 to 60 (arbitrary units). The obtained experimental mass fragmentation spectra were collected and compared to in silico mass fragmentation profiles of each marker, simulated with Mass Frontier software (v. 6.0; Thermo Scientific, San Jose, CA).
Intending to confirm our findings, tandem mass spectrometry (MS/MS) was applied in the same instrument mentioned above. The collision gas used was helium, with collision-induced dissociation energy ranging from 30 to 60 (arbitrary units). The obtained experimental mass fragmentation spectra were collected and compared to in silico mass fragmentation profiles of each marker, simulated with Mass Frontier software (v. 6.0; Thermo Scientific, San Jose, CA).