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GNPS - UM Matthaei botanical garden & Herbarium (low m/z)

ABSTRACT: Plant metabolic extracts. Plant tissues as specified in file name were collected from adult live plants in the University of Michigan Matthaei Botanical Garden. 0.2g fresh weight tissue were frozen, ground in a Tissuelyzer for 2min at 6m/s with 10 2.3mm silica beads in 2mL vials. Ground tissue was extracted with 3mL methanol for 1h at 37 celsius shaking. Methanol was separated from insoluble material and dried under nitrogen. Dried crude methanol extracts were resuspended in water, partitioned twice with 3 mL hexane, 3 mL ethyl acetate and finally extracted with 3 mL n-butanol. Ethylacetate and n-butanol extracts were dried in a speedvac concentrator, resuspended in 1 mL methanol, filtered through a 0.2 micron filter and analyzed by LC-MS/MS on an Thermo QExactive orbitrap with a ESI source. LC-MS settings were as follows: Injection volume 5 uL, LC - Phenomenex Kinetex 2.6 um C18 reverse phase 100 A 150 x 3 mm LC column, LC gradient: solvent A - 0.1% formic acid, solvent B - acetonitrile (0.1% formic acid), 0 min: 10% B, 5 min: min: 60% B, 5.1 min: 95% B, 6 min: 95% B, 6.1 min: 10% B, 9.9 min: 10% B, 0.5 mL/min, MS - positive ion mode, Full MS: Resolution 70000, mass range 150-450 m/z, dd-MS2 (data-dependent MS/MS): resolution 17500, AGC target 1e5, Loop count 5, Isolation width 1.0 m/z, Collision energy 25 eV, dynamic exclusion 0.5 s.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Viridiplantae (ncbitaxon:33090)

SUBMITTER: Desnor Chigumba

PROVIDER: MSV000092820 | GNPS |

REPOSITORIES: GNPS

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Project description:All chemicals were from Fisher Scientific unless otherwise noted. All solvents for extraction were HPLC grade, all solvents for mass spectrometry analysis were LCMS (Thermo Optima) grade. Streptomyces ansochromogenes subspecies pallens NRRL B-12018 was obtained from the ARS Culture Collection of the United States Department of Agriculture. A 30 mL Streptomyces ansochromogenes subspecies pallens NRRL B-12018 was inoculated from an ISP2 agar culture and cultivated in liquid ISP2 medium (4 g yeast extract, 10 g malt extract, 4 g D-glucose in 1 L deionized water, pH 7.3) for 11 days at 30 celsius at 225 rpm. The medium was then centrifuged in 50 mL centrifuge tube at 3200 g for 30 min. The cell pellet and supernatant were separated subsequently, and the supernatant (30 mL) was extracted three times with 50 mL ethyl acetate to remove lipophilic components. Then the supernatant was dried by rotary evaporation at 40 celsius. Finally, the dried supernatant was resuspended in 3.5 mL deionized water, centrifuged for 5 min at 16000 g, filtered with a Cytiva syringeless filter (0.2 micron mesh size, Cytiva, US503NPEORG). The filtered supernatant was immediately analyzed by liquid-chromatography-tandem mass spectrometry on a Thermo QExactive orbitrap mass spectrometer with a H-ESI-II ion source coupled to a Vanquish HPLC system. The LC parameters were as follows: Phenomenex Kinetex 2.6 micrometer C18 reverse phase 100 Ang. 150 x 3 mm LC column, solvent A: 20mM ammonium formate (pH 3.2), solvent B: 90% acetonitrile and 10% 200 mM ammonium formate (pH 3.2), column compartment temperature 30 celsius, injection volume 5 microliter, flow rate 0.5 mL/min, 0 min: 0% B, 5 min: 40% B, 4.5 min: 95% B, 5.5 min: 95% B, 6 min: 0% B, 10 min: 0% B. Electrospray ion source (Thermo H-ESI-II source) parameters were as follows: negative ion mode, sheath gas flow 35 psi, aux gas flow 3 psi, electrospray capillary temperature 320 celsius, electrospray capillary voltage 3.6 kV. MS parameters were as follows: full MS, resolution 70000, mass range 400-1200 m/z, dd-MS2 (data-dependent MS/MS), resolution 17500, AGC target 1x10^5, loop count 5, isolation width 1.0 m/z, collision energy 25 eV and dynamic exclusion 0.5 s. LC-MS data were analyzed with QualBrowser in the Thermo Xcalibur software package (v.4.3.73.11, Thermo Scientific).

Project description:For core peptide analysis, enzyme assays were performed using 50 uL volumes containing either copper(II) chloride (CuCl2) from JT Baker Chemical Co. or water. Assays for BURP-domain proteins comprised 300 uM GheBURP-1xQLFVWGW/100 uM ManBURP-1xQLFFWRY, 1 mM CuCl2, and a citrate:phosphate buffer (pH 7) with final concentrations of 18 mM citrate and 165 mM Na2HPO4. The enzyme reactions were incubated for 24 h at 20 C. Subsequently, 1 ug of trypsin (Sigma-Aldrich, T7575-1KT) in 20 uL of 1 M Tris-HCl (pH 8)/1 ug of LysC (NEB, P8109S) in 10 uL of 10 mM Tris-HCl (pH 8) buffer was added to GheBURP/ManBURP assay, respectively, and further incubated at 37 C for 24 h. Each digest was centrifuged, added to ChromacolTM LC-MS vial, and subjected to LC-MS/MS analysis. LC-MS/MS settings: Injection volume 15 uL, LC - Higgins Analytical PROTO300 C4 5 um 250 x 4.6 mm, solvent A - 0.1% formic acid, solvent B - acetonitrile (0.1% formic acid), 0.7 mL/min, LC gradient: 0 min: 10% B, 10 min: 50% B, 11 min: 95% B, 13 min: 95% B, 13.1 min: 10% B, 20min: 10% B, MS - Positive ion mode, Full MS: Resolution 35000, mass range: 700-1250 m/z for GheBURP digest and 700-1450 m/z for ManBURP digest, dd-MS2: resolution 17500, AGC target 1e5, loop count 5, isolation window 0.4 m/z, collision energy 25 eV, dynamic exclusion 2 s. Scaled enzyme assays for exoproteolytic cyclic peptide formation were conducted on a 2 mL scale using purified GheBURP-1xQLFVWGW/ManBURP-1xQLFFWRY obtained from a 4 L/2 L expression and purification process. These assays included 130-350 uM of the BURP domain cyclase and 1 mM CuCl2 in a citrate:phosphate buffer at pH 7. The assays were incubated for 24 h at 20 C. Following incubation, trypsin/LysC was introduced into the GheBURP/ManBURP reaction mixtures at a protease-substrate molar ratio of 1:250/1:100, with the pH adjusted to 8 using a 1 M Tris-HCl (pH 8) buffer and incubated at 37 C for 24 h. Two digests were subjected to preparative HPLC, respectively (LC settings, Phenomenex Kinetex 5 um C18 100 A LC Column 150 x 21.2 mm, 7.5 mL/min; solvent A, 0.1% TFA; solvent B, acetonitrile (0.1% TFA); LC gradient, 0 min, 10% B; 1 min, 10% B; 36 min, 50% B; 39 min, 95% B; 42 min, 95% B; 42.5 min, 10% B; 60.1 min, 10% B). HPLC fractions were analyzed by LC-MS analysis for target masses of modified core peptides with the following LC-MS parameters: injection volume 2 uL; LC, Phenomenex Kinetex 2.6 um C18 reverse phase 100 A 50 x 3 mm LC column; LC gradient. solvent A, 0.1% formic acid; solvent B, acetonitrile (0.1% formic acid); 0 min, 5% B; 2.5 min, 95% B; 3.0 min, 95% B; 3.1 min, 5% B; 5.0 min, 5% B, 0.5 mL/min; MS, positive ion mode; full MS, resolution 35000, mass range 400-1200 m/z; dd-MS2, resolution 17500; loop count 5, collision energy 25 eV and dynamic exclusion 0.5 s. LC fractions containing target peptides were dried and resuspended in 30 uL DMSO for further analysis. Exopeptidase assays were conducted with approximately 25-50 ug of peptides (with 4% DMSO), carboxypeptidase Y (from S. cerevisiae, Sigma-Aldrich), and aminopeptidase N (from Rat, Sigma Aldrich) at a peptidase:peptides ratio of 1:10 (w/w). These assays were performed in 100 mM Tris-HCl buffer at pH 7 and 37 C for 24 h. Following incubation, the assays were centrifuged and analyzed by LC-MS/MS as follows: Injection volume 15 uL, LC - Higgins Analytical PROTO300 C4 5 um 250 x 4.6 mm, all gradients: solvent A - 0.1% formic acid, solvent B - acetonitrile (0.1% formic acid), 0.7 mL/min, LC gradient: 0 min: 15% B, 60 min: 40% B, 61 min: 95% B, 63 min: 95% B, 63.1 min: 15% B, 80 min: 15% B, MS - Positive ion mode, Full MS: Resolution 35000, mass range: 800-1300 m/z for GheBURP and 700-1450 m/z for ManBURP, dd-MS2: resolution 17500, AGC target 1e5, loop count 5, isolation width 0.4 m/z, collision energy 25 eV, dynamic exclusion 0.6 s. The analysis was compared to purified glechomanin/mercurialin standards.

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GNPS - UM Matthaei botanical garden & Herbarium (low m/z)

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