Journal Article

A Proteomic Approach to Identification of Transmembrane Proteins and Membrane-anchored Proteins of <i>Arabidopsis thaliana</i> by Peptide Sequencing

Takashi Sazuka, Sumie Keta, Katsuhiro Shiratake, Shohei Yamaki and Daisuke Shibata

in DNA Research

Published on behalf of Kazusa DNA Research Institute

Volume 11, issue 2, pages 101-113
Published in print January 2004 | ISSN: 1340-2838
Published online January 2004 | e-ISSN: 1756-1663 | DOI: http://dx.doi.org/10.1093/dnares/11.2.101

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A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved e.ective for the identification of membrane-bound proteins.

Keywords: proteomics; membrane-bound protein; transmembrane protein; membrane-anchored protein; Arabidopsis thaliana

Journal Article.  0 words. 

Subjects: Genetics and Genomics

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