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Data‐directed top‐down Fourier‐transform mass spectrometry of a large integral membrane protein complex: Photosystem II from Galdieria sulphuraria
Author(s) -
Thangaraj Balakumar,
Ryan Christopher M.,
Souda Puneet,
Krause Kirsten,
Faull Kym F.,
Weber Andreas P. M.,
Fromme Petra,
Whitelegge Julian P.
Publication year - 2010
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.201000190
Subject(s) - mass spectrometry , photosystem ii , fourier transform ion cyclotron resonance , chemistry , protein mass spectrometry , top down proteomics , tandem mass spectrometry , analytical chemistry (journal) , electron capture dissociation , chromatography , biochemistry , photosynthesis
High‐resolution top‐down MS was used to characterize eleven integral and five peripheral subunits of the 750 kDa photosystem II complex from the eukaryotic red alga, Galdieria sulphuraria . The primary separation used LC MS with concomitant fraction collection (LC‐MS+), yielding around 40 intact mass tags at 100 ppm mass accuracy on a low‐resolution ESI mass spectrometer, whose retention and mass were used to guide subsequent high‐resolution top‐down nano‐electrospray FT ion‐cyclotron resonance MS experiments (FT‐MS). Both collisionally activated and electron capture dissociation were used to confirm the presence of eleven small subunits to mass accuracy within 5 ppm; PsbE, PsbF, PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, PsbT, PsbX and PsbZ. All subunits showed covalent modifications that fall into three classes including retention of initiating formyl‐methionine, removal of methionine at the N ‐terminus with or without acetylation, and removal of a longer N ‐terminal peptide. Peripheral subunits identified by top‐down analysis included oxygen‐evolving complex subunits PsbO, PsbU, PsbV, as well as Psb28 (PsbW) and Psb27 (“PsbZ‐like”). Top‐down high‐resolution MS provides the necessary precision, typically less than 5 ppm, for identification and characterization of polypeptide composition of these important membrane protein complexes.