12 t magnet

For top-down
fragmentation with electron capture dissociation, protein samples
were prepared in 100 mM ammonium acetate and ionized using a Triversa
Nanomate as described above. Spectra were acquired using a SolariX
FT-ICR 2XR instrument equipped with a 12 T magnet (Bruker Daltonics).
Prior to fragmentation, individual protein charge states were isolated
in the mass resolving quadrupole. Ions were accumulated for up to
500 ms in the ICR cell in order to typically achieve a signal of around
10⁸ per scan. ECD cathode conditions were a bias of 1.7
V and a lens voltage of 20 V for monomeric protein and a bias of 1.5
V and a lens voltage of 22 V for the dimeric protein. Typically, an
ECD pulse length of between 5 and 15 ms was used.

Sample preparation was carried out as above for three biological replicates of each wild type, Δnog1 and ΔcitC mutants. Metabolite profiling was conducted using Ion cyclotron resonance Fourier transform Mass spectrometry (ICR-FT/MS) on a Bruker solariX equipped with a 12 T magnet (Bruker Daltonics, Bremen). Putative metabolites were annotated using the MassTRIX webserver [34 (link)]. Statistical analysis was carried out in MS Excel 2010 and Genedata Expressionist for MS 7.6 (Genedata, Martinsried) using Welch’s T test (p ≤ 0.05) [35 , 36 (link)].

Plasma samples from a total of 376 subjects (see above for details and ethical approvals), from two independent cohorts, were analyzed via DI-FT-ICR MS (24 (link), 41 (link)). Before analyses, the metabolites (from 50 μl of blood plasma) were extracted by C18 solid-phase extraction (SPE) technology, using Omix C18 100 μl tips (Varian) and following the protocol described by Forcisi et al. (24 (link)). The extracts, diluted in methanol by a factor of 50, were analyzed in positive ESI mode via DI-FT-ICR MS, using a Bruker SolariX instrument equipped with a 12-T magnet (Bruker Daltonik GmbH, Bremen, Germany). The instrument was externally calibrated by injecting a solution of arginine (10 μg/ml) and observing corresponding peaks with m/z values equal to 175.11895 [M+H]⁺, 349.23062 [2M+H]⁺, 523.34230 [3M+H]⁺, and 697.45397 [4M+H]⁺. In the experiment, the flow rate of infusion was set to 120 μl/hour. Four hundred scans, each corresponding to 4 MWs in the interval from 147.4 to 1000.0 m/z, were acquired and averaged. The time of accumulation ion was set to 0.7 s, and the time of flight to the detector was set to 1 ms. The voltages of capillary and spray shields were set to 3800 and −500 V, respectively. The flow rate of nebulizer gas was kept at 2.2 bar, and the drying gas flow rate was set to 4 liters/min (at a temperature of 180°C).