Journal Article

Absolute quantification of <i>Medicago truncatula</i> sucrose synthase isoforms and N-metabolism enzymes in symbiotic root nodules and the detection of novel nodule phosphoproteins by mass spectrometry

Stefanie Wienkoop, Estíbaliz Larrainzar, Mirko Glinski, Esther M. González, Cesar Arrese-Igor and Wolfram Weckwerth

in Journal of Experimental Botany

Published on behalf of Society for Experimental Biology

Volume 59, issue 12, pages 3307-3315
Published in print September 2008 | ISSN: 0022-0957
Published online September 2008 | e-ISSN: 1460-2431 | DOI: http://dx.doi.org/10.1093/jxb/ern182

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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), 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 3.2.1.26) and an RNA-binding protein.

Keywords: Absolute quantification; alkaline invertase; asparagine synthetase; de novo sequencing; mass western; Medicago truncatula; phosphoproteomics; plant proteomics; root nodules; stable isotope labelling; sucrose synthase

Journal Article.  4689 words.  Illustrated.

Subjects: Plant Sciences and Forestry

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