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Submitted on August 7, 2002
Revised on October 5, 2002
Accepted on October 11, 2002

Full subunit coverage liquid chromatography electrospray-ionization mass spectrometry (LCMS+) of an oligomeric membrane protein: Cytochrome b6f complex from spinach and the cyanobacterium, M. laminosus

Julian P. Whitelegge, Huamin Zhang, Rodrigo Aguilera, Ross Taylor, and William A. Cramer

Chemistry, UCLA, Los Angeles, CA 90095

Corresponding Author: jpw{at}chem.ucla.edu

Highly active cytochrome b6f complexes from spinach and the cyanobacterium M. laminosus have been analyzed by liquid-chromatography with electrospray-ionization mass spectrometry (LCMS+). Both size-exclusion and reverse-phase separations were used to separate protein subunits allowing measurement of their molecular weights to an accuracy exceeding 0.01 % (+/- 3Da at 30000 Da). The products of petA, petB, petC, petD, petG, petL, petM and petN were detected in complexes from both spinach and M. laminosus while the spinach complex also contained ferredoxin-NADP+ oxidoreductase (FNR; J. Biol. Chem. 276, 38159-38165). While the measured masses of PetC and PetD (18935.8 and 17311.8 Da, respectively) from spinach are consistent with the published primary structure, the measured masses of cytochrome f (31934.7 Da, PetA) and cytochrome b (24886.9 Da, PetB) are in modest deviation from values calculated based upon genomic sequence and known post-translational modifications. The low molecular weight protein subunits have been sequenced using tandem mass spectrometry (MSMS) without prior cleavage. Sequences derived from the MSMS spectra of these intact membrane proteins in the range 3.2 to 4.2 kDa were compared to translations of genomic DNA sequence, where available. Products of the spinach chloroplast genome, PetG, PetL and PetN, all retained their initiating formyl methionine while the nuclear encoded PetM was cleaved after import from the cytoplasm. While the sequences of PetG and PetN revealed no discrepancy with translations of the spinach chloroplast genome, Phe was detected at position 2 of PetL. The spinach chloroplast genome reports a codon for Ser at position 2 implying the presence of a DNA sequencing error or a previously undiscovered RNA-editing event. Clearly, complete annotation of genomic data requires detailed expression measurements of primary structure by mass spectrometry. Full subunit coverage of an oligomeric intrinsic membrane protein complex by LCMS+ presents a new facet to intact mass proteomics.


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