Analysis of root plasma membrane aquaporins from brassica oleracea: Post-translational modifications, de novo sequencing and detection of isoforms by high resolution mass spectrometry Articles uri icon

authors

  • CASADO VELA, JUAN
  • Muries, B.
  • Iloro, I.
  • Carvajal, M.
  • Elortza, F.
  • Martínez-Ballesta, M.C.

publication date

  • January 2010

start page

  • 3479

end page

  • 3494

issue

  • 7

volume

  • 9

International Standard Serial Number (ISSN)

  • 1535-3893

Electronic International Standard Serial Number (EISSN)

  • 1535-3907

abstract

  • Plasma membrane Intrinsic Proteins (PIPs), a subfamily of aquaporins, are ubiquitous membrane channel proteins that play a crucial role in water uptake in plants. The use of high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) analysis of peptides has previously shown to be a valuable tool to differentiate among PIP homologues sharing a high sequence homology and also to characterize their post-translational modifications (PTMs). The recent introduction of mass spectrometers able to measure peptide mass with high mass accuracy, together with new alternative ways of peptide fragmentation allows the identification and characterization of proteins from nonsequenced organisms, such as broccoli. In this study, we combined three endoproteases (trypsin, Glu-C and Lys-C) with HPLC-MS/MS analysis and two types of peptide fragmentations, CID (collision induced dissociation) and HCD (higher-energy C-trap dissociation), to identify PIP isoforms and PTMs from broccoli roots. After de novo sequencing analysis, eight peptides showing homology to Arabidopsis thaliana PIPs were identified. Although Arabidopsis nomenclature of PIP isoforms has not been defined for broccoli, our results agree with the occurrence of seven AtPIP isoforms (PIP 1;1, PIP 1;2, PIP 1;3 and PIP2;2, PIP 2;3, PIP2;1 and PIP2;7) in broccoli roots, as compared to the plant model A. thaliana. To our knowledge, these results represent the deepest characterization of the PIPs isolated from the roots of broccoli, a crop with increasing agronomical interest. © 2010 American Chemical Society.

keywords

  • brassica oleracea de novo sequencing ltq orbitrap plasma membrane intrinsic protein post-translational modifications aquaporin isoprotein lysine trypsin aquaporin isoprotein vegetable protein accuracy arabidopsis article broccoli cabbage cell membrane collisionally activated dissociation high performance liquid chromatography mass spectrometry methodology nonhuman priority journal protein analysis protein processing sequence analysis sequence homology water transport acetylation amino acid sequence brassica chemistry methylation molecular genetics nucleotide sequence phosphorylation plant root procedures sequence alignment tandem mass spectrometry arabidopsis arabidopsis thaliana brassica oleracea brassica oleracea var. italica pips acetylation amino acid sequence aquaporins brassica cell membrane chromatography; high pressure liquid conserved sequence methylation molecular sequence data phosphorylation plant proteins plant roots protein isoforms protein processing; post-translational sequence alignment sequence analysis; protein tandem mass spectrometry