Tsq altis mass spectrometer

All the MRM experiments were carried out on TSQ Altis mass spectrometer (ThermoFisher Scientific, USA) coupled to a Vanquish uHPLC (ThermoFisher Scientific, USA) platform. 1µg of peptides was loaded on to a Hypersil Gold C18 column 1.9μm 100 X 2.1mm (ThermoFisher Scientific, USA) and chromatographic separation of peptides was carried out at a flow rate of 0.40ml/min for CSF samples and 0.45ml/min for tissue samples. The total time of gradient for CSF samples was 20 minutes, while that for tissue samples was 10 minutes. The buffer system was binary with Buffer A (0.1% Formic acid in water) and Buffer B (80% Acetonitrile in 0.1% Formic acid water). The gradients used have been shown in Supplementary Table 2. With an ESI source to the MS, the data was acquired for 20 minutes in case of CSF samples and 10 minutes in case of tissue samples. All the other MS relevant parameters (which were kept similar for both kinds of samples) are tabulated in Supplementary Figure 1.

Mice were treated with 120 mg/kg 5‐ALA via intraperitoneal injection 1 h prior to sacrifice. Brain and tumor samples were snap‐frozen in liquid nitrogen and homogenized in ice cold methanol:acetonitrile:water (2:2:1, v/v/v). Metabolite extracts were analyzed by reversed phase chromatography directly coupled to mass spectrometry (LC–MS/MS). For each sample, 1 μl was injected onto a Kinetex (Phenomenex) C18 column (100 Å, 150 × 2.1 mm) connected with a respective guard column, employing a 3‐min‐long linear gradient from 3% A (1% acetonitrile, 0.1% formic acid in water) to 95% B (0.1% formic acid in acetonitrile) at a flow rate of 80 μl/min, followed by isocratic elution for 25 min. Detection and quantification were done by selected reaction monitoring (SRM), employing a TSQ Altis mass spectrometer (Thermo Fisher Scientific) and using the following transitions in the positive ion mode: 210–122 m/z (porphobilinogen), 211–163 m/z, 563–504 m/z (protoporphyrin IX). Data interpretation was performed using Xcalibur (Thermo Fisher Scientific). Authentic standards were used for determining the optimal collision energies and for the confirmation of experimental retention times via standards added to the control sample.

Acyl-ACPs were measured by following the method described by Nam et al. (2020) (link). Briefly, fresh leaf tissues were harvested from 5- to 6-wk-old Arabidopsis plants and quickly frozen. Frozen tissues were ground to a fine powder using liquid nitrogen and a mortar and pestle and weighed into individual tubes, taking care throughout the weighing process to avoid thawing. The tissue was homogenized in a solution of 5% (w/v) trichloroacetic acid (TCA). Precipitated proteins were pelleted by centrifugation, washed with 1% (w/v) TCA, and resuspended in MOPS buffer (50 mm, pH 7.6). Proteins were precipitated again by adding TCA to a concentration of 10% (w/v), followed by centrifugation and washing of the pellet with 1% (w/v) TCA. Precipitated proteins were then resuspended in 50 µL of MOPS buffer and digested with Endoproteinase AspN (Cat# P3303, MilliporeSigma, Waltham, MA, USA). Digestions were quenched with the addition of 50 µL methanol, centrifuged at 15,000 × g, and acyl-ACPs in the supernatant analyzed using an Ultimate 3000 liquid chromatography system connected to a TSQ Altis mass spectrometer from Thermo Scientific (MilliporeSigma, Waltham, MA, USA) (see parameters in Supplemental Method S1) or AB Sciex QTRAP 6500 (Framingham, MA, USA) with settings, production of acyl-ACP standards, and more extensive protocol details as described in Jenkins et al. (2021) (link).

Detection and quantification of amino acids and dipeptides was done by LC–MS/MS, using a Vanquish HPLC system coupled to a TSQ Altis mass spectrometer (both Thermo Fisher Scientific), employing the Selected Reaction Monitoring (SRM) mode and positive polarity. In brief, dried samples were resolved in 0.1% formic acid in water and 1 µL was injected onto a Kinetex (Phenomenex) C18 column (100 Å, 150 × 2.1 mm), employing a 9-min-long linear gradient from 100% A (1% acetonitrile, 0.1% formic acid in water) to 90% B (0.1% formic acid in acetonitrile) at a flow rate of 100 µL/min. Retention times, SRM transitions, and optimal collisional energies were determined by authentic standards and degenerate dipeptide libraries. All data interpretation was performed using Xcalibur (Thermo Fisher Scientific).

The data was acquired using a TSQ Altis Mass Spectrometer (ThermoFisher Scientific) connected with an HPLC system (Dionex Ultimate 3000 -ThermoFisher Scientific). Peptides were separated using Hypersil Gold C18 column (1.9 μm, 100 × 2.1 mm, ThermoFisher Scientific). MRM runs were performed using a flow rate of 450 µL/min, a cycle time of 2 s, and a resolution of 0.7 m/z (Q1 and Q3) over an LC gradient of 10 min. The solvent system included 0.1% FA and 100% Acetonitrile (ACN). The data obtained was further analyzed using Skyline daily where peak selection and refinement were made based on considering the peak shape, dot product and retention time. For further statistical analysis, the values for peak area were exported, and peptide-wise comparison was made.

Targeted liquid chromatography-tandem mass spectrometry triple quadrupole (LC-QQQ) analysis was performed on a TSQ Altis mass spectrometer (Thermo Scientific, Waltham, MA) coupled to a binary pump UHPLC (Vanquish, Thermo Scientific). Scan parameters for target ions were pravastatin—polarity negative, precursor m/z 423, products m/z 101, 303, and 321; The injection volume was 10 µL. Chromatographic separation was achieved on a Hypersil Gold 5 μm, 50 mm × 2.1 mm C18 column (Thermo Scientific) maintained at 30 °C using a solvent gradient method. Solvent A was 0.1% formic acid in water. Solvent B was 0.1% formic acid in acetonitrile. The gradient method used was 0–1 min (20% B to 60% B), 1–2 min (60% B to 95% B), 2–4 min (95% B), 4–4.1 min (95% B to 20% B), 4.1–5 min (20% B). The flow rate was 0.5 mL min-1. Sample acquisition and data analysis were performed Trace Finder 4.1 (Thermo Scientific).

A Vanquish™ Flex LC pump interfaced with a TSQ Altis mass spectrometer (MS) (Thermo Scientific™, San Jose, CA, USA) was used to analyze all analytes. The column used for all analyses was a Hypersil GOLD™ aQ Vanquish 50 × 2.1, 1.9 µm (Thermo Scientific™). Mobile Phase A was water plus 0.1% formic acid, and Mobile Phase B was acetonitrile plus 0.1% formic acid for all analyses. The column compartment temperature was 40 °C for all analyses. The chromatographic gradient was as follows: start 0% organic, linear ramp from 0.5 to 2 min up to 80% organic (100% organic for propranolol), hold at 80% organic (100% organic for propranolol) for 0.4 min, return to starting conditions for 0.01 min, and hold at starting conditions for 0.59 min. The solvent flow rate was 0.4 mL/min. A 2 µL injection volume was used.
The following MS method parameters were used for the analysis of all analytes. The resolution of Q1 and Q3 was 0.7 FWHM. The CID gas was set to 1.5 mTorr. The chromatographic peak width was 2 s, and the cycle time was 0.2 s. The source conditions were as follows: positive mode spray voltage 2000 V, sheath gas 60 Arb, auxiliary gas 22 Arb, sweep gas 1 Arb, ion transfer tube temperature 350 °C, and vaporizer temperature 350 °C. The total run time of the method was 3 min.