title: Turnover rates in microorganisms by laser ablation electrospray ionization mass spectrometry and pulse-chase analysis.
abstract: Biochemical processes rely on elaborate networks containing thousands of compounds participating in thousands of reaction. Rapid turnover of diverse metabolites and lipids in an organism is an essential part of homeostasis. It affects energy production and storage, two important processes utilized in bioengineering. Conventional approaches to simultaneously quantify a large number of turnover rates in biological systems are currently not feasible. Here we show that pulse-chase analysis followed by laser ablation electrospray ionization mass spectrometry (LAESI-MS) enable the simultaneous and rapid determination of metabolic turnover rates. The incorporation of ion mobility separation (IMS) allowed an additional dimension of analysis, i.e., the detection and identification of isotopologs based on their collision cross sections. We demonstrated these capabilities by determining metabolite, lipid, and peptide turnover in the photosynthetic green algae, Chlamydomonas reinhardtii, in the presence of (15)N-labeled ammonium chloride as the main nitrogen source. Following the reversal of isotope patterns in the chase phase by LAESI-IMS-MS revealed the turnover rates and half-lives for biochemical species with a wide range of natural concentrations, e.g., chlorophyll metabolites, lipids, and peptides. For example, the half-lives of lyso-DGTS(16:0) and DGTS(18:3/16:0), t1/2 = 43.6 ± 4.5 h and 47.6 ± 2.2 h, respectively, provided insight into lipid synthesis and degradation in this organism. Within the same experiment, half-lives for chlorophyll a, t1/2 = 24.1 ± 2.2 h, and a 2.8 kDa peptide, t1/2 = 10.4 ± 3.6 h, were also determined.
authors.affiliation: Department of Chemistry, W.M. Keck Institute for Proteomics Technology and Applications, The George Washington University, Washington, DC 20052, USA.
authors.forename: Sylwia A
authors.initials: SA
authors.lastname: Stopka
authors.affiliation: Department of Chemistry, W.M. Keck Institute for Proteomics Technology and Applications, The George Washington University, Washington, DC 20052, USA.
authors.forename: Tarek R
authors.initials: TR
authors.lastname: Mansour
authors.affiliation: Department of Chemistry, W.M. Keck Institute for Proteomics Technology and Applications, The George Washington University, Washington, DC 20052, USA.
authors.forename: Bindesh
authors.initials: B
authors.lastname: Shrestha
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168, CEA-CNRS-INRA-Univ. Grenoble Alpes, Grenoble, France.
authors.forename: Éric
authors.initials: É
authors.lastname: Maréchal
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168, CEA-CNRS-INRA-Univ. Grenoble Alpes, Grenoble, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Falconet
authors.affiliation: Department of Chemistry, W.M. Keck Institute for Proteomics Technology and Applications, The George Washington University, Washington, DC 20052, USA. Electronic address: vertes@gwu.edu.
authors.forename: Akos
authors.initials: A
authors.lastname: Vertes
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D007553:Isotope Labeling; D007834:Lasers; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
keywords: Cell; Laser ablation electrospray ionization; Mass spectrometry; Pulse-chase analysis; Turnover rate