a-simple-fractionated-extraction-method-for-comprehensive-analysis

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Max Planck Institute of Molecular Plant Physiology, Golm, Germany

Germination and early seedling establishment are developmental stages in which plants face limited nutrient supply as their photosynthesis mechanism is not yet active. For this reason, the plant must mobilize the nutrient reserves provided by the mother plant in order to facilitate growth. Autophagy is a catabolic process enabling the bulk degradation of cellular constituents in the vacuole. The autophagy mechanism is conserved among eukaryotes, and homologs of many autophagy-related (ATG) genes have been found in Arabidopsis thaliana. T-DNA insertion mutants (atg mutants) of these genes display higher sensitivity to various stresses, particularly nutrient starvation. However, the direct impact of autophagy on cellular metabolism has not been well studied. In this work, we used etiolated Arabidopsis seedlings as a model system for carbon starvation. atg mutant seedlings display delayed growth in response to carbon starvation compared with wild-type seedlings. High-throughput metabolomic, lipidomic, and proteomic analyses were performed, as well as extensive flux analyses, in order to decipher the underlying causes of the phenotype. Significant differences between atg mutants and wild-type plants have been demonstrated, suggesting global effects of autophagy on central metabolism during carbon starvation as well as severe energy deprivation, resulting in a morphological phenotype.

Global analysis of the role of autophagy in cellular metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation.

The elucidation of complex biological systems requires integration of multiple molecular parameters. Accordingly, high throughput methods like transcriptomics, proteomics, metabolomics and lipidomics have emerged to provide the tools for successful system-wide investigations. Unfortunately, optimized analysis of different compounds requires specific extraction procedures in combination with specific analytical instrumentation. However, the most efficient extraction protocols often only cover a restricted number of compounds due to the different physico-chemical properties of these biological compounds. Consequently, comprehensive analysis of several molecular components like polar primary metabolites next to lipids or proteins require multiple aliquots to enable the specific extraction procedures required to cover these diverse compound classes. This multi-parallel sample handling of different sample aliquots is therefore not only more sample intensive, it also requires more time and effort to obtain the required extracts.

Protocol: a fast, comprehensive and reproducible one-step extraction method for the rapid preparation of polar and semi-polar metabolites, lipids, proteins, starch and cell wall polymers from a single sample.

Cyclophilins (CYPs) are a group of ubiquitous proteins characterized by their ability to bind to the immunosuppressive drug cyclosporin A. The CYP family occurs in a wide range of organisms and contains a conserved peptidyl-prolyl cis/trans isomerase domain. In addition to fulfilling a basic role in protein folding, CYPs may also play diverse important roles, e.g. in protein degradation, mRNA processing, development, and stress responses. We performed a genome-wide database survey and identified a total of 94 CYP genes encoding 91 distinct proteins. Sequence alignment analysis of the putative BnCYP cyclophilin-like domains revealed highly conserved motifs. By using RNA-Seq, we could verify the presence of 77 BnCYP genes under control conditions. To identify phloem-specific BnCYP proteins in a complementary approach, we used LC-MS/MS to determine protein abundances in leaf and phloem extracts. We detected 26 BnCYPs in total with 12 being unique to phloem sap. Our analysis provides the basis for future studies concentrating on the functional characterization of individual members of this gene family in a plant of dual importance: as a crop and a model system for polyploidization and long-distance signalling.

Article	Year
Global analysis of the role of autophagy in cellular metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation. The Plant cell\| PubMed ID: 25649436	2015
Protocol: a fast, comprehensive and reproducible one-step extraction method for the rapid preparation of polar and semi-polar metabolites, lipids, proteins, starch and cell wall polymers fro... Plant methods\| PubMed ID: 27833650	2016
Bioinformatic and expression analysis of the Brassica napus L. cyclophilins. Scientific reports\| PubMed ID: 28473712	2017

Krzysztof Bajdzienko

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