TY - JOUR
T1 - Data-directed top-down Fourier-transform mass spectrometry of a large integral membrane protein complex
T2 - Photosystem II from Galdieria sulphuraria
AU - Thangaraj, Balakumar
AU - Ryan, Christopher M.
AU - Souda, Puneet
AU - Krause, Kirsten
AU - Faull, Kym F.
AU - Weber, Andreas P M
AU - Fromme, Petra
AU - Whitelegge, Julian P.
PY - 2010/10
Y1 - 2010/10
N2 - 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 100ppm mass accuracy on a low-resolution ESI mass spectrometer, whose retention and mass were used to guide subsequent high-resolution topdown 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.
AB - 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 100ppm mass accuracy on a low-resolution ESI mass spectrometer, whose retention and mass were used to guide subsequent high-resolution topdown 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.
KW - FT-MS
KW - Membrane proteins
KW - Photosystem II
KW - Red algae
KW - Technology
KW - Top-down proteomics
UR - http://www.scopus.com/inward/record.url?scp=78649436428&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=78649436428&partnerID=8YFLogxK
U2 - 10.1002/pmic.201000190
DO - 10.1002/pmic.201000190
M3 - Article
C2 - 20845333
AN - SCOPUS:78649436428
SN - 1615-9853
VL - 10
SP - 3644
EP - 3656
JO - Proteomics
JF - Proteomics
IS - 20
ER -