Dataset Information

GNPS Glasgow Polyomics Metabolite Standards (mainly amino acid, sugar, nucleotide, and TCA cycle metabolism) measured with pHILIC and HILIC gradients

ABSTRACT: This dataset contains full scan and fragmentation data for pHILIC-MS and HILIC-MS gradients of more than 150 standards run at Glasgow Polyomics for metabolite identification and annotation purposes - separated over 3 batches to accommodate for solubility, co-elution, and isomer effects. Where available, the results of a targeted search for metabolites from the respective standard mixtures were added. Full lists of there three mixtures are also added in the data entry. All relevant chromatography and mass spectrometry details are provided below. Please acknowledge Glasgow Polyomics if you found this metabolite standard data of relevance for your study results. pHILIC and HILIC chromatography used: The samples were analysed using a Thermo Scientific Ultimate 3000 RSLCnano system (Thermo Scientific, CA, USA). The pHILIC separation was performed with a SeQuant ZIC-pHILIC column (150 x 4.6 mm, 5 microm) equipped with the corresponding pre-column (Merck KGaA, Darmstadt, Germany) - the column temperature was maintained at 25 C. A linear biphasic LC gradient was conducted from 80% B to 20% B over 15 min, followed by a 2 min wash with 5% B, and 8 min re-equilibration with 80% B, where solvent B is acetonitrile and solvent A is 20 mM ammonium carbonate in water. The flow rate was 300 microL/min, column temperature was held at 25 C, injection volume was 10 microL, and samples were maintained at 4 C in the autosampler. HILIC chromatography was as above but with a same sized ZIC-HILIC column and a linear gradient from 80% B to 20% B over 30 min, followed by an 8 min wash with 5% B, and 8 min re-equilibration with 80% B, where solvent B is 0.08% formic acid in acetonitrile and solvent A is 0.1% formic acid in water. MS equipment and settings used: The LC system was coupled to a Thermo Scientific Q-Exactive Orbitrap mass spectrometer equipped with a HESI II interface (Thermo Scientific, Hemel Hempstead, UK). The set up was calibrated (Thermo calmix) in both ionization modes and tuned for the lower m/z range before analysis. Full scan (MS1) data was acquired in positive and negative switching mode in profile mode at 35,000 resolution (at m/z 200) using 1 microscan, an AGC target of 106 cts, a maximum injection time of 250 ms, with spray voltages +3.8 and -3.0 kV, capillary temperature 320 C, heater temperature of 150 C, sheath gas flow rate 40 a.u., auxiliary gas flow rate 5 a.u., sweep gas flow rate 5 a.u, a full scan mass window of 70-1050 m/z, and using m/z 74.0964 (+) and m/z 112.98563 (-) as locking masses. Fragmentation data (LC-MS/MS) was obtained in positive and negative ionization combined and separate fragmentation modes as described in (van der Hooft et al 2016). Briefly, for separate mode, a duty cycle consisted of one full scan (MS1) event and one Top5 (or Top10) MS/MS (MS2) fragmentation event, with full scan (MS1) resolution (at m/z 200) was set to 70,000, the AGC target set to 1 x 106, and the maximum injection time set to 120 ms. MS/MS (MS2) resolution (at m/z 200) was set to 17,500, the AGC target set to 2 x 105, MS/MS maximum injection time was set to 80 ms and the underfill ratio was set to 10 %, with a resulting intensity threshold of 2.5 × 105 cts. For combined mode, a duty cycle consisted of two of the above events in positive and negative ionization mode with the following modifications: full scan (MS1) resolution (at m/z 200) was set to 35,000, MS/MS resolution was set to 35,000, the AGC target was set to 1 x 105, and the maximum MS/MS filling time was set to 120 ms with an underfill ratio of 20%, resulting in an intensity threshold of 1.7 x 105. Further settings were as specified for full scan analysis above.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Metabolite Standards

SUBMITTER: Justin van der Hooft

PROVIDER: MSV000082661 | GNPS | Wed Jul 25 04:48:00 BST 2018

REPOSITORIES: GNPS

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Project description:Total lysates were prepared from IRF3 KO 293T cells transfected with Flag-IRF3 and followed by immunoprecipitation with α-FLAG M2 beads. Immunoprecipitated proteins were separated by SDS-PAGE, and then stained with Coomassie Blue. The entire lane was excised, digested with trypsin, and analyzed with LC-MS/MS. LC-MS/MS identification of peptide mixtures was performed once with two replicates per condition at BIOPROFILE TECHNOLOGY (Shanghai, China). Briefly, peptides were chromatographed through Easy-nLC1200 (Thermo Fisher, California, USA). Peptide samples were loaded by Trap column (reverse-phase), (100 μm*2 cm, 5μm, C18, Dr. Maisch GmbH) and separated by Thermo scientific EASY column (75 μm*15cm, 3 μm, C18) at 300 nL min−1 for 60 min using a four-step acetonitrile (0.1% formic acid in 80% acetonitrile) gradient: 2–5% over the first 2 min and 5–28% for 2–44 min and 28–40% for 44–51 min and 40–100% for 51–53 min. The tandem mass spectrometry was performed by Q Exactive mass spectrometer (Thermo Fisher, California, USA). The MS1 survey scan (350–1800 m/z) was at a resolution of 60,000 at 200 m/z with automatic gain control (AGC) target of 3e6 and a maximum injection time of 50 ms. Dynamic exclusion was 60.0 s. Each full scan takes 20 MS2 scans. MS2 activation type was HCD model. Isolation window was 2 m/z. The MS2 survey was at a resolution of 15,000 at 200 m/z with automatic gain control (AGC) target of 1e5 and a maximum injection time of 50 ms. RAW files generated by spectrometer was subjected to Proteome Discoverer (version 1.4) software with searching the library of Uniprot homo sapiens (uniprot-Homo sapiens (Human) [9606]-204995-20220606.fasta) for protein identification. Trypsin was specified as the proteolytic enzyme, with up to two missed cleavage sites allowed. Carbamidomethylation was set as the fixed modification. Oxidation (M) and ubiquitination [GlyGly (K)] were set as the variable modifications. The precursor mass tolerance was set to 20 ppm and the fragment ion tolerance at 0.1 Da. Proteins were identified based on at least one unique peptide. Protein and peptide identification confidence threshold were set to an FDR of 1%. The tandem mass spectra of matched peptides were checked manually for their validity.

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Dataset Information

GNPS Glasgow Polyomics Metabolite Standards (mainly amino acid, sugar, nucleotide, and TCA cycle metabolism) measured with pHILIC and HILIC gradients

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