title: Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry.
abstract: The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam, Germany. giavalisco@mpimp-golm.mpg.de
authors.forename: Patrick
authors.initials: P
authors.lastname: Giavalisco
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Annemarie
authors.initials: A
authors.lastname: Matthes
authors.forename: Aenne
authors.initials: A
authors.lastname: Eckhardt
authors.forename: Hans-Michael
authors.initials: HM
authors.lastname: Hubberten
authors.forename: Holger
authors.initials: H
authors.lastname: Hesse
authors.forename: Shruthi
authors.initials: S
authors.lastname: Segu
authors.forename: Jan
authors.initials: J
authors.lastname: Hummel
authors.forename: Karin
authors.initials: K
authors.lastname: Köhl
authors.forename: Lothar
authors.initials: L
authors.lastname: Willmitzer
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism*; D002247:Carbon Isotopes; D002734:Chlorophyll / Q000031:analogs & derivatives / Q000737:chemistry; D016208:Databases, Factual; D005583:Fourier Analysis; D007553:Isotope Labeling; D008055:Lipids / Q000032:analysis; D013058:Mass Spectrometry; D055432:Metabolomics / Q000379:methods*; D009587:Nitrogen Isotopes; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification*; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism; D018517:Plant Roots / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013460:Sulfur Isotopes
chemical_list: D002247:Carbon Isotopes; D008055:Lipids; D009587:Nitrogen Isotopes; D010936:Plant Extracts; D013460:Sulfur Isotopes; D002734:Chlorophyll; C032623:pheophorbide a