Full subunit coverage liquid chromatography electrospray ionization mass spectrometry (LCMS+) of an oligomeric membrane protein: cytochrome b(6)f complex from spinach and the cyanobacterium Mastigocladus laminosus.
Highly active cytochrome b(6)f complexes from spinach and the cyanobacterium Mastigocladus 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 masses to an accuracy exceeding 0.01% (+/-3 Da at 30,000 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 (Zhang, H., Whitelegge, J. P., and Cramer, W. A. (2001) Flavonucleotide:ferredoxin reductase is a subunit of the plant cytochrome b(6)f complex. 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) modestly deviate 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 of 3.2-4.2 kDa were compared with translations of genomic DNA sequence where available. Products of the spinach chloroplast genome, PetG, PetL, and PetN, all retained their initiating formylmethionine, 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.
Whitelegge
Julian P
JP
The Pasarow Mass Spectrometry Laboratory, Department of Psychiatry, Neuropsychiatric Institute, University of California, Los Angeles, California 90095, USA. jpw@chem.ucla.edu
Zhang
Huamin
H
Aguilera
Rodrigo
R
Taylor
Ross M
RM
Cramer
William A
WA
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D000458:Cyanobacteria / Q000378:metabolism*; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism*; D019143:Evolution, Molecular; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D018724:Spinacia oleracea / Q000378:metabolism*; D020524:Thylakoids / Q000737:chemistry
D003573:Cytochrome b Group; D008565:Membrane Proteins; D009842:Oligopeptides