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Multidimensional proteomic analysis of photosynthetic membrane proteins by liquid extraction-ultracentrifugation-liquid chromatography-mass spectrometry

Huber, Christian G. and Walcher, Wolfgang and Timperio, Anna Maria and Troiani, Sonia and Porceddu, Andrea and Zolla, Lello (2004) Multidimensional proteomic analysis of photosynthetic membrane proteins by liquid extraction-ultracentrifugation-liquid chromatography-mass spectrometry. Proteomics, Vol. 4 (12), p. 3909-3920. eISSN 1615-9861. Article.

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DOI: 10.1002/pmic.200400823

Abstract

The membrane protein components of photosystem I (PSI) and II (PSII) from different species were prefractionated by liquid extraction and sucrose gradient ultracentrifugation and subsequently analyzed by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) using poly-(styrene-divinylbenzene)-based monolithic capillary columns. The analytical method was shown to be very flexible and enabled the identification of antenna proteins as well as most of the proteins of the reaction center from PSI and PSII in various plant species with few RP-HPLC-ESI-MS analyses necessitating only minor adaptations in the gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid. The membrane proteins, ranging in molecular mass (Mr) from 4196 (I protein) to more than 80 000 (PSI A/B) as well as isoforms were identified on the basis of their intact Mr and comparison with Mr deduced from known DNA or protein sequences. High quality mass spectra enabled the identification and quantitation of the nonphosphorylated and phosphorylated reaction center subunits D1, D2, and CP43 of PSII, containing five to seven membrane-spanning -helices. Because of its high flexibility and suitability for proteins having a very wide range of Mr and hydrophobicities, the method is generally applicable to the analysis of complex mixtures of membrane proteins.

Item Type:Article
ID Code:3979
Status:Published
Refereed:Yes
Uncontrolled Keywords:Chloroplast, electrospray ionization mass spectrometry, liquid chromatography, membrane proteins, photosystem
Subjects:Area 07 - Scienze agrarie e veterinarie > AGR/07 Genetica agraria
Divisions:001 Università di Sassari > 01 Dipartimenti > Scienze agronomiche e genetica vegetale agraria
Publisher:Wiley
eISSN:1615-9861
Deposited On:27 May 2010 12:20

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