Fibroblast PI Incorporation and MMUT Activity

PI into acid-precipitable material of primary fibroblasts was assessed according to a protocol described previously^{42 (link)} with modifications as described²⁰. MMUT enzyme activity assay was performed in fibroblast crude cell lysates as originally described^{43 ,44 (link)} using recent modifications^{8 (link)}. MMUT enzyme activity in HEK cells was measured using the same protocol but without radiolabeled substrate (instead only 1 mM of methylmalonyl-CoA was used, Sigma M1762) and final succinate determination was performed by HPLC separation and electrospray ionization (ESI) tandem mass spectrometry (MS/MS) detection (SCIEX TripleQuad 5500 LC–MS/MS System).

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Forny P., Bonilla X., Lamparter D., Shao W., Plessl T., Frei C., Bingisser A., Goetze S., van Drogen A., Harshman K., Pedrioli P.G., Howald C., Poms M., Traversi F., Bürer C., Cherkaoui S., Morscher R.J., Simmons L., Forny M., Xenarios I., Aebersold R., Zamboni N., Rätsch G., Dermitzakis E.T., Wollscheid B., Baumgartner M.R, & Froese D.S. (2023). Integrated multi-omics reveals anaplerotic rewiring in methylmalonyl-CoA mutase deficiency. Nature Metabolism, 5(1), 80-95.

Publication 2023

methylmalonyl-CoA M1762 TripleQuad 5500 LC–MS/MS System

Acid Cells Electrospray ionization mass spectrometry Enzyme activity Enzyme assay Fibroblast cell Hplc Mass spectrometry Methylmalonyl coa Succinate

Corresponding Organization :

Other organizations : University Children's Hospital Zurich, University of Zurich, ETH Zurich, SIB Swiss Institute of Bioinformatics

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Variable analysis

independent variables

Protocol described previously with modifications

dependent variables

PI into acid-precipitable material of primary fibroblasts
MMUT enzyme activity in fibroblast crude cell lysates
MMUT enzyme activity in HEK cells

control variables

Control variables not explicitly mentioned

controls

Positive control: Not specified
Negative control: Not specified

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