Linear ion trap mass spectrometer

Nonbiased metabolomic screening of whole blood and plasma of wild type (WT) mice and SCD mice (n = 6 for each group) was performed using LC/GC-MS as described previously30 (link)51 (link). Specifically, a Thermo Fisher linear ion-trap mass spectrometer with Fourier transform and a Mat-95 XP mass spectrometer were used to analyze 7,000 named metabolites. The LC/MS POS platform was optimized for compounds that show positive ionization as described previously51 (link). There were 251 small metabolites detected in the plasma and whole blood of both the controls and SCD mice. The combinations of groups were analyzed using Welch’s 2-sample t test, following log transformation and imputation with minimum observed values for each compound. P < 0.05 was considered significant. q-value was used as a measure of “false discovery rate (FDR)”. A FDR of 0.05 means that on average 5% of the truly null features in the study will be called significant52 (link).

Manipulations were performed as we reported recently11 (link). Protein components in the LD preparation were precipitated with 100% acetone. Proteins were separated by 10% SDS-PAGE followed by Coomassie Blue or silver staining. For the total proteome, a full lane of Coomassie Blue-stained gel was cut into 23 slices from high to low molecular weight. Each slice was further cut into smaller pieces, destained, washed, dehydrated and vacuum-dried. Proteins in slices were reduced with 10 mM dithiothreitol for 1 h at 56 °C and alkylated with 55 mM iodoacetamide for 45 min. Gel slices were washed with 25 mM ammonium bicarbonate, acetonitrile and vacuum-dried. For in-gel digestion, slices were incubated with 10 ng/μl trypsin in 25 mM ammonium bicarbonate solution. The digestion reaction proceeded at 37 °C overnight and was stopped by adding 5% formic acid to adjust pH to <4.0. After two extractions with 60% acetonitrile, the tryptic peptide mixture was vacuum-dried and dissolved in 0.1% formic acid. Peptide extracts were purified on a C18 trap column and analyzed by use of a 2D-HPLC system coupled to a linear ion-trap mass spectrometer (Thermo Fisher Scientific, MA).

After permethylation, structural analysis of N-glycans was performed in a linear ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA) in positive ion mode. Glycans dissolved in methanol were loaded at 0.5 µL/min at a sheath gas flow rate of 2 arb, a spray voltage of 3.50 kV, a capillary voltage of 35 V, a capillary temperature of 230 °C, a tube lens of 120 V, an injection time of 100 ms, an activation time of 30 ms, an activation Q-value of 0.250, and an isolation width of m/z 1.5. To further identify the structure of N-glycans, MSⁿ spectra were obtained. All samples were analyzed as sodium-adducted positive ions.