TY - BOOK

T1 - Lanthanide Elements as Labels in Multiplexed Analysis of Proteins and MicroRNAs by Laser Ablation Inductively Coupled Plasma Mass Spectrometry

AU - de Bang, Thomas Christian

PY - 2014

Y1 - 2014

N2 - Analytical methods for targeted and multiplexed analysis of proteins and nucleic acids (DNA and RNA) are important tools for investigating how environmental stimuli affect biological entities at the molecular level. Specific analyses of proteins and nucleic acids can be achieved on the basis of a wide variety of different analytical techniques, each with a number of advantages and disadvantages. In this PhD study, two bioanalytical assays were developed for the specific detection of nine thylakoid proteins and three microRNAs. Despite the different types of analytes, the basic principles of the two assays were very similar. Protein or RNA samples were separated by polyacrylamide gel electrophoresis and transferred to membranes by electroblotting where specific recognition of target analytes was achieved by antibodies and DNA probes, respectively. The use of lanthanides as labels on different antibodies and DNA probes, enabled quantitative and multiplexed analysis of the analytes using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The results obtained by the new methods were compared to different state-of-the-art techniques and the analytical figures of merits were similar. The developed methodology was used to analyse the effect of manganese deficiency on the composition of selected photosystem II subunits in barley (Hordeum vulgare L.). As an effect of manganese deficiency, the abundances of the extrinsic proteins PsbP and PsbQ decreased, and it was shown that this dynamic was manganese dependent.

AB - Analytical methods for targeted and multiplexed analysis of proteins and nucleic acids (DNA and RNA) are important tools for investigating how environmental stimuli affect biological entities at the molecular level. Specific analyses of proteins and nucleic acids can be achieved on the basis of a wide variety of different analytical techniques, each with a number of advantages and disadvantages. In this PhD study, two bioanalytical assays were developed for the specific detection of nine thylakoid proteins and three microRNAs. Despite the different types of analytes, the basic principles of the two assays were very similar. Protein or RNA samples were separated by polyacrylamide gel electrophoresis and transferred to membranes by electroblotting where specific recognition of target analytes was achieved by antibodies and DNA probes, respectively. The use of lanthanides as labels on different antibodies and DNA probes, enabled quantitative and multiplexed analysis of the analytes using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The results obtained by the new methods were compared to different state-of-the-art techniques and the analytical figures of merits were similar. The developed methodology was used to analyse the effect of manganese deficiency on the composition of selected photosystem II subunits in barley (Hordeum vulgare L.). As an effect of manganese deficiency, the abundances of the extrinsic proteins PsbP and PsbQ decreased, and it was shown that this dynamic was manganese dependent.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122138862005763

M3 - Ph.D. thesis

BT - Lanthanide Elements as Labels in Multiplexed Analysis of Proteins and MicroRNAs by Laser Ablation Inductively Coupled Plasma Mass Spectrometry

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -