LC-MS/MS-Massenspektrometer mit linearer Ionenfalle

• Subject Area: Plant Sciences
• Term: Funded in 2007
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 50971395

• Final Report Year: 2011

Final Report Abstract

The LTQ-Orbitrap mass spectrometer was employed to continue our research aimed to understand the regulation of photosynthesis in response to various stresses in the green alga Chlamydomonas reinhardtii. For this purpose we further improved comparative proteomics approaches talking advantage of metabolic labeling strategies. We previously used comparative proteomics to identify candidate proteins involved in the response to iron-deficiency. In this regard we characterized the function of LHCSR3 and demonstrated that this protein is essential for energydependent non-photochemical quenching. To this end we employed mass spectrometry to characterize the content of light-harvesting proteins in wild-type and LHCSR3-deficient mutants. Comparative proteome analyses using stable isotope labeling of amino acids revealed insights into the anaerobic response of the green alga. The focus of the work was put on the chloroplast and more than 400 chloroplast proteins could be quantitated. In addition this work defined a core chloroplast proteome of C. reinhardtii consisting of 996 proteins . These localization data will provide an important basis for further functional studies and modeling purposes. Candidate anaerobic response genes that were identified by the comparative proteomics approach were investigated by reverse genetics employing RNAi and amiRNA approaches. These studies identified TEF7 as a new player in the anaerobic response since mutant strains having lower levels of this protein cannot survive under anaerobic growth conditions. Using comparative and absolute mass spectrometric quantitation, the composition of photosystem I and its associated light-harvesting proteins from the green alga C. reinhardtii and from the unicellular red alga Cyanidioschyzon merolae was investigated. In the course of the proteomic experiments, a new mass spectrometric evaluation pipeline for peptide and protein identification and quantitation was established and the mass spectrometric data were additionally utilized for a proteogenomic approach serving for improved annotation of the C. reinhardtii gene models. In an additional research line we established phospho-proteomics to map in vivo and in vitro protein phosphorylation sites using SIMAC fractionation (sequential elution from IMAC) and identification of the peptides and proteins by mass spectrometry using the MultiStage activation modus. This approach was utilized to determine phosphoproteins in the process of flagellar disassembly during flagellar shortening in Chlamydomonas. Furthermore we are engaged in the analysis of calcium signaling via protein phosphorylation in plant model cell types during environmental stress adaptation.

Publications

• (2008). 2DB: a Proteomics database for storage, analysis, presentation, and retrieval of information from mass spectrometric experiments. BMC Bioinformatics 9: 302
  Allmer J, Kuhlgert S, Hippler M
  
• (2008). Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photo-oxidative stress in response to iron availability in Chlamydomonas reinhardtii. Plant J 55: 201-211
  Busch A, Rimbauld B, Naumann B, Rensch S, Hippler M
  
• (2009). An ancient light-harvesting protein is critical for the regulation of algal photosynthesis. Nature 462: 518-521
  Peers G, Truong TB, Ostendorf E, Busch A, Elrad D, Grossman AR, Hippler M, Niyogi KK
  
• (2009). Proteotypic profiling of LHCI from Chlamydomonas reinhardtii provides new insights into structure and function of the complex. Proteomics 9: 398-408
  Stauber EJ, Busch A, Naumann B, Svatos A, Hippler M
  
• (2010). A novel replicative enzyme encoded by the linear Arthrobacter plasmid pAL1. J Bacteriol 192: 4935-4943
  Kolkenbrock S, Naumann B, Hippler M, Fetzner S
  
• (2010). Characterizing the Anaerobic Response of Chlamydomonas reinhardtii by Quantitative Proteomics. Mol Cell Proteomics 9: 1514-1532
  Terashima M, Specht M, Naumann B, Hippler M
  
• (2010). The composition and structure of photosystem I-associated antenna from Cyanidioschyzon merolae. Plant J 62: 886-897
  Busch A, Nield J, Hippler M
  
• (2011). Concerted action of the new Genomic Peptide Finder and AUGUSTUS allows for automated proteogenomic annotation of the Chlamydomonas reinhardtii genome. Proteomics 11: 1814-1823
  Specht M, Stanke M, Terashima M, Naumann-Busch B, Janssen I, Hohner R, Hom EF, Liang C, Hippler M
  
• (2011). Protein Phosphorylation Is a Key Event of Flagellar Disassembly Revealed by Analysis of Flagellar Phosphoproteins during Flagellar Shortening in Chlamydomonas. Journal of proteome research 10: 3830-3839
  Pan J, Naumann-Busch B, Wang L, Specht M, Scholz M, Trompelt K, Hippler M
  
• (2011). Proteomics to go: Proteomatic enables the user-friendly creation of versatile MS/MS data evaluation workflows. Bioinformatics 27: 1183-1184
  Specht M, Kuhlgert S, Fufezan C, Hippler M