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Multidimensional proteomic analysis of photosynthetic membrane proteins by liquid extraction‐ultracentrifugation‐liquid chromatography‐mass spectrometry
Author(s) -
Huber Christian G.,
Walcher Wolfgang,
Timperio AnnaMaria,
Troiani Sonia,
Porceddu Andrea,
Zolla Lello
Publication year - 2004
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200400823
Subject(s) - chemistry , chromatography , mass spectrometry , electrospray ionization , extraction (chemistry) , membrane , thylakoid , biochemistry , chloroplast , gene
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 ( M r ) from 4196 (I protein) to more than 80 000 (PSI A/B) as well as isoforms were identified on the basis of their intact M r and comparison with M r 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 α‐helices. Because of its high flexibility and suitability for proteins having a very wide range of M r and hydrophobicities, the method is generally applicable to the analysis of complex mixtures of membrane proteins.