© 2008 The Author(s).
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RESEARCH PAPER |
Absolute quantification of Medicago truncatula sucrose synthase isoforms and N-metabolism enzymes in symbiotic root nodules and the detection of novel nodule phosphoproteins by mass spectrometry
1Institute of Biochemistry and Biology, University of Potsdam, c/o MPI-MP, D-14476 Potsdam, Germany
2Departamento de Ciencias del Medio Natural, Edifico de los Olivos, Universidad Pública de Navarra, E-31006 Pamplona, Navarra, Spain
3Agronomy Physiology Laboratory, Department of Agronomy, University of Florida, POB 110965, Gainesville, FL 32611, USA
* To whom correspondence should be addressed. E-mail: wienkoop{at}mpimp-golm.mpg.de
Mass spectrometry (MS) has become increasingly important for tissue specific protein quantification at the isoform level, as well as for the analysis of protein post-translational regulation mechanisms and turnover rates. Thanks to the development of high accuracy mass spectrometers, peptide sequencing without prior knowledge of the amino acid sequence—de novo sequencing—can be performed. In this work, absolute quantification of a set of key enzymes involved in carbon and nitrogen metabolism in Medicago truncatula ‘Jemalong A17’ root nodules is presented. Among them, sucrose synthase (SuSy; EC 2.4.1.13 [EC] ), one of the central enzymes in sucrose cleavage in root nodules, has been further characterized and the relative phosphorylation state of the three most abundant isoforms has been quantified. De novo sequencing provided sequence information of a so far unidentified peptide, most probably belonging to SuSy2, the second most abundant isoform in M. truncatula root nodules. TiO2-phosphopeptide enrichment led to the identification of not only a phosphorylation site at Ser11 in SuSy1, but also of several novel phosphorylation sites present in other root nodule proteins such as alkaline invertase (AI; EC 3.2.1.2 [EC] 6) and an RNA-binding protein.
Key words: Absolute quantification, alkaline invertase, asparagine synthetase, de novo sequencing, mass western, Medicago truncatula, phosphoproteomics, plant proteomics, root nodules, stable isotope labelling, sucrose synthase
Received 12 April 2008; Revised 17 June 2008 Accepted 18 June 2008