Metabolite Extraction from Tissue Samples

Small molecule metabolites were extracted as described previously [17 (link)] with some modifications. Biopsy samples were resuspended in ice-cold 60% aqueous methanol and homogenized using a tissue homogenizer (Tissue Tearor Model 985370–395, Biospec Products Inc., Bartlesville, OK) set to 2-minute intervals of 10 seconds on and 5 seconds off. Tissues were transferred to glass tubes and lysed by sonication prior to addition of 1:1 aqueous choloroform and vortexing. Aqueous layers were collected by centrifugation and transferred to clean tubes prior to lyophilization for 4 hours with low heat. Lyophilized samples were resuspended in 550 μL of NMR buffer (10mM NaH₂PO₄/ Na₂HPO₄ containing 0.5 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) in 100% D₂O, pH 7), assayed for pH, and transferred to 5mm NMR tubes (Bruker) prior to analysis.

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Ammons M.C., Morrissey K., Tripet B.P., Van Leuven J.T., Han A., Lazarus G.S., Zenilman J.M., Stewart P.S., James G.A, & Copié V. (2015). Biochemical Association of Metabolic Profile and Microbiome in Chronic Pressure Ulcer Wounds. PLoS ONE, 10(5), e0126735.

Publication 2015

5mm NMR tubes

Biopsy Buffer Centrifugation Cold Lyophilization Methanol Seconds Sulfonic acid Tissues

Corresponding Organization :

Other organizations : Montana State University, University of Montana, Johns Hopkins University

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Protocol cited in 4 other protocols

Identifying Rhomboid Protease Cleavage Sites

To determine the cleavage site of rhomboid substrates, HEK293T cells were transfected as described above, lysed in
RIPA buffer, and subjected to anti-FLAG immunopurification (Sigma). Eluent was spotted onto a sinapinic acid matrix and
analyzed by MALDI-TOF mass spectrometry on a standards-calibrated Voyager DE Instrument (AB SCIEX) as previously described
(Moin and Urban, 2012 (link)). Resultant spectra were analyzed and plotted in the R
environment with aid of the MALDIquant package (Gibb and Strimmer, 2012).

Gandhi S., Baker R.P., Cho S., Stanchev S., Strisovsky K, & Urban S. (2020). Designed parasite-selective rhomboid inhibitors block invasion and clear blood-stage malaria. Cell chemical biology, 27(11), 1410-1424.e6.

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Purification and Mass Spectrometry of Proteolytic Products

Full-length substrate and C-terminal cleavage products were purified from in vitro proteolysis assays by anti-Flag immunoaffinity isolation and analyzed by MALDI-TOF mass spectrometry using sinapinic acid matrix as described previously^{19 (link),32}.

Baker R.P, & Urban S. (2015). Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating. Nature, 523(7558), 101-105.

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Purification and Mass Spectrometry of Proteolytic Products

Baker R.P, & Urban S. (2015). Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating. Nature, 523(7558), 101-105.

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Membrane Modulation of HEK293T Cells

HEK293T cells (ATCC, Manassas, USA), which are neuronal in origin (Shaw et al., 2002 (link)), were transfected using X-tremeGENE HP (Roche, Basel, Switzerland), washed 22 hours post-transfection, and incubated in serum-free media containing membrane-altering agents (0.004% lyso-phospholipids or 2.5mM methyl-β-cyclodextrin) or NSAIDs (0.5mM each except 0.1mM for sulindac sulfide, or as specified otherwise) for ~18–24 hours. Cells were lysed in Laemmli buffer, resolved on 4–20% Tris-Glycine SDS-PAGE gels, and subjected to quantitative western analysis using infrared fluorescence (LiCor Biosciences, Lincoln, USA). Cell viability was assessed using trypan blue exclusion. MALDI-TOF mass spectrometry was performed as described (Moin and Urban, 2012 (link)).

Urban S, & Moin S.M. (2014). A subset of membrane-altering agents and γ-secretase modulators provoke non-substrate cleavage by rhomboid proteases. Cell reports, 8(5), 1241-1247.

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Variable analysis

independent variables

Modifications to the previously described [17] metabolite extraction protocol

dependent variables

Small molecule metabolites

control variables

Homogenization using a tissue homogenizer with set intervals of 10 seconds on and 5 seconds off
Lyophilization for 4 hours with low heat
Resuspension in NMR buffer (10mM NaH2PO4/Na2HPO4 containing 0.5 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) in 100% D2O, pH 7)

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