title: Multiplexed quantification of plant thylakoid proteins on Western blots using lanthanide-labeled antibodies and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS).
abstract: We have developed a novel calibration method that allows concurrent quantification of multiple proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) after Western blotting. Calibrants were made of nitrocellulose membranes doped with lanthanide standards. Excellent linearity was obtained in the interval from 0 to 24 ng lanthanide cm(-2). Cerium-labeled lysozyme was introduced as an internal reference protein, enabling correction for up to 50% difference in transfer efficiency during the blotting of membranes. The sensitivity of the LA-ICP-MS method was comparable to state-of-the-art chemiluminescence detection and was further improved by a factor of 20, using a polymer tag. Our method allowed reproducible and multiplexed quantification of five thylakoid proteins extracted from chloroplasts of the plant species Arabidopsis thaliana (relative standard deviation (RSD) of ≤ 5% in three independent analytical series). The method was capable of measuring the L subunit in photosystem I of an Arabidopsis mutant containing <5% of this particular protein, relative to the wild type. We conclude that the developed calibration method is highly suited for multiplexed and comparative protein studies, allowing for intermembrane comparisons with high sensitivity and reproducibility.
authors.affiliation: Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark.
authors.forename: Thomas Christian
authors.initials: TC
authors.lastname: de Bang
authors.forename: Pai
authors.initials: P
authors.lastname: Pedas
authors.forename: Jan Kofod
authors.initials: JK
authors.lastname: Schjoerring
authors.forename: Poul Erik
authors.initials: PE
authors.lastname: Jensen
authors.forename: Søren
authors.initials: S
authors.lastname: Husted
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D029681:Arabidopsis Proteins / Q000032:analysis*; D015153:Blotting, Western / Q000379:methods*; D002138:Calibration; D002563:Cerium / Q000737:chemistry*; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D010720:Phosphines / Q000737:chemistry; D013194:Staining and Labeling; D020524:Thylakoids / Q000737:chemistry*
chemical_list: D000906:Antibodies; D029681:Arabidopsis Proteins; D010720:Phosphines; D002563:Cerium