Whitelegge Julian P, Zhang Huamin, Aguilera Rodrigo, Taylor Ross M, Cramer William A
The Pasarow Mass Spectrometry Laboratory, Department of Psychiatry, Neuropsychiatric Institute, University of California, Los Angeles, California 90095, USA.
Mol Cell Proteomics. 2002 Oct;1(10):816-27. doi: 10.1074/mcp.m200045-mcp200.
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.
利用电喷雾电离质谱法（LCMS+）对菠菜和蓝细菌层理鞭枝藻中高活性的细胞色素b(6)f复合物进行了分析。采用尺寸排阻和反相分离法分离蛋白质亚基，从而能够精确测定其分子量，精度超过0.01%（30,000 Da时为±3 Da）。在菠菜和层理鞭枝藻的复合物中均检测到了petA、petB、petC、petD、petG、petL、petM和petN的产物，而菠菜复合物中还含有铁氧还蛋白-NADP(+)氧化还原酶（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）。虽然菠菜中PetC和PetD的实测分子量（分别为18935.8和17311.8 Da）与已发表的一级结构一致，但细胞色素f（31934.7 Da，PetA）和细胞色素b（24886.9 Da，PetB）的实测分子量与根据基因组序列和已知翻译后修饰计算的值略有偏差。利用串联质谱法（MSMS）对低分子量蛋白质亚基进行了测序，无需事先裂解。将这些完整膜蛋白在3.2 - 4.2 kDa范围内的MSMS谱图所得序列与可用的基因组DNA序列翻译结果进行了比较。菠菜叶绿体基因组的产物PetG、PetL和PetN均保留了起始甲酰甲硫氨酸，而核编码的PetM在从细胞质导入后被裂解。虽然PetG和PetN的序列与菠菜叶绿体基因组的翻译结果没有差异，但在PetL的第2位检测到苯丙氨酸。菠菜叶绿体基因组报告第2位的密码子为丝氨酸，这意味着存在DNA测序错误或先前未发现的RNA编辑事件。显然，基因组数据的完整注释需要通过质谱法对一级结构进行详细的表达测量。通过LCMS+对寡聚内在膜蛋白复合物进行全亚基覆盖，为完整质量蛋白质组学展现了一个新的方面。
