title: Analysis of low abundant trehalose-6-phosphate and related metabolites in Medicago truncatula by hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry.
abstract: Trehalose-6-phosphate (T6P) is an important signaling metabolite involved in plant growth control that inhibits the sucrose nonfermenting-1-related protein kinase 1 (SnRK1), a key regulator of energy and carbon metabolism in plants. The quantification of T6P in plant tissues is fundamental to improve our understanding of sugar signaling and the links between plant growth and development in response to stress conditions. However, the almost undetectable levels of T6P together with the complex plant matrix and the presence of T6P isomers such as sucrose-6-phosphate (S6P), makes the detection of this metabolite challenging. This work describes the development and validation of a hydrophilic interaction chromatography (HILIC) method for the on-line coupling with negative ion electrospray (ESI) triple quadrupole tandem mass spectrometry (MS/MS) in the highly sensitive and selective multiple reaction monitoring (MRM) mode for the target analysis of metabolic intermediates of the biosynthesis of trehalose, including glucose-6-phosphate (G6P), uridine 5-diphospho-glucose (UDPG), T6P (and its isomer S6P). Enhanced signal in the MRM mode and improved chromatographic separation for each compound were obtained using piperidine and methylphosphonic acid as additives in the HILIC mobile phase. The optimized HILIC-ESI-QqQ-MS/MS method increases the range of sensitive analytical methodologies for the quantification of key low-abundant metabolites, and was applied to quantify the fluctuations of S6P, T6P and G6P in Medicago truncatula plants in response to environmental stress. The levels of S6P, T6P, and G6P in M. truncatula plant tissues (roots and leaves) exposed to a water deficit and recovery treatment, ranged from 30 to 150pmolg-1FW, 16-120pmolg-1FW, and 330-1690pmolg-1FW, respectively.
authors.affiliation: Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Av. República, 2780-157 Oeiras, Portugal.
authors.forename: Ana Teresa
authors.initials: AT
authors.lastname: Mata
authors.affiliation: Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Av. República, 2780-157 Oeiras, Portugal.
authors.forename: Tiago Filipe
authors.initials: TF
authors.lastname: Jorge
authors.affiliation: iMED, Faculdade de Farmácia da Universidade de Lisboa (FFUL), Av. Prof. Gama Pinto, 1649-019 Lisboa, Portugal.
authors.forename: João
authors.initials: J
authors.lastname: Ferreira
authors.affiliation: iMED, Faculdade de Farmácia da Universidade de Lisboa (FFUL), Av. Prof. Gama Pinto, 1649-019 Lisboa, Portugal; Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Analytical Chemistry Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Maria
authors.initials: M
authors.lastname: do Rosário Bronze
authors.affiliation: Plant Cell Biotechnology Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Branco
authors.affiliation: Plant Cell Biotechnology Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Av. da República, 2780-157 Oeiras, Portugal; Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa (FCUL), Campo Grande, 1749-016 Lisboa, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Fevereiro
authors.affiliation: Plant Cell Biotechnology Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Av. da República, 2780-157 Oeiras, Portugal; Plant Biotechnology Laboratory, Department of Biology and Biotechnology 'L. Spallanzani', University of Pavia, via Ferrata 9, 27100, Pavia, Italy.
authors.forename: Susana
authors.initials: S
authors.lastname: Araújo
authors.affiliation: Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Av. República, 2780-157 Oeiras, Portugal. Electronic address: antonio@itqb.unl.pt.
authors.forename: Carla
authors.initials: C
authors.lastname: António
mesh_terms: D053898:Biosynthetic Pathways; D002853:Chromatography, Liquid / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D057230:Limit of Detection; D046913:Medicago truncatula / Q000378:metabolism*; D055442:Metabolome; D018515:Plant Leaves / Q000378:metabolism; D012015:Reference Standards; D012996:Solutions; D013395:Sucrose / Q000031:analogs & derivatives / Q000378:metabolism; D013403:Sugar Phosphates / Q000032:analysis* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014199:Trehalose / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: D012996:Solutions; D013403:Sugar Phosphates; C535046:sucrose-6-phosphate; C082722:trehalose-6-phosphate; D013395:Sucrose; D014199:Trehalose
keywords: HILIC-ESI-QqQ-MS/MS; Medicago truncatula; Method validation; Quantitative MS; Trehalose-6-phosphate; Water deficit