title: A laser ablation ICP-MS based method for multiplexed immunoblot analysis: applications to manganese-dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.).
abstract: Manganese (Mn) constitutes an essential co-factor in the oxygen-evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody-based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods, based on data-dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re-supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn-dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA-ICP-MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA-ICP-MS provides an analytical platform with high throughput appropriate for screening large collections of plants.
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark.
authors.forename: Thomas Christian
authors.identifier: http://orcid.org/0000-0002-5019-6422
authors.initials: TC
authors.lastname: de Bang
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark, Denmark.
authors.forename: Jørgen
authors.initials: J
authors.lastname: Petersen
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark.
authors.forename: Pai Rosager
authors.initials: PR
authors.lastname: Pedas
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark, Denmark.
authors.forename: Adelina
authors.initials: A
authors.lastname: Rogowska-Wrzesinska
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark, Denmark.
authors.forename: Ole Noerregaard
authors.initials: ON
authors.lastname: Jensen
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark.
authors.forename: Jan Kofod
authors.initials: JK
authors.lastname: Schjoerring
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark.
authors.forename: Poul Erik
authors.initials: PE
authors.lastname: Jensen
authors.affiliation: Christopher S. Bond Life Sciences Center, University of Missouri, 1201 Rollins St., Columbia, MO 65211, USA.
authors.forename: Jay J
authors.initials: JJ
authors.lastname: Thelen
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Husted
mesh_terms: D001467:Hordeum / Q000378:metabolism*; D015151:Immunoblotting; D007834:Lasers / Q000706:statistics & numerical data*; D008345:Manganese / Q000378:metabolism*; D045332:Photosystem II Protein Complex / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D045332:Photosystem II Protein Complex; C054266:photosystem II manganese-stabilizing protein; D008345:Manganese
keywords: PsbO; PsbP; PsbQ; barley (Hordeum vulgare L.); data-dependent acquisition; extrinsic proteins; laser ablation inductively coupled plasma mass spectrometry; manganese deficiency; photosystem II; selected reaction monitoring; technical advance