Forbes Andrew J, Patrie Steven M, Taylor Gregory K, Kim Yong-Bin, Jiang Lihua, Kelleher Neil L
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2678-83. doi: 10.1073/pnas.0306575101. Epub 2004 Feb 19.
For more complete characterization of DNA-predicted proteins (including their posttranslational modifications) a "top-down" approach using high-resolution tandem MS is forwarded here by its application to methanogens in both hypothesis-driven and discovery modes, with the latter dependent on new automation benchmarks for intact proteins. With proteins isolated from ribosomes and whole-cell lysates of Methanococcus jannaschii (approximately 1,800 genes) using a 2D protein fractionation method, 72 gene products were identified and characterized with 100% sequence coverage via automated fragmentation of intact protein ions in a custom quadrupole/Fourier transform hybrid mass spectrometer. Three incorrect start sites and two modifications were found, with one of each determined for MJ0556, a 20-kDa protein with an unknown methylation at approximately 50% occupancy in stationary phase cells. The separation approach combined with the quadrupole/Fourier transform hybrid mass spectrometer allowed targeted and efficient comparison of histones from M. jannaschii, Methanosarcina acetivorans (largest Archaeal genome, 5.8 Mb), and yeast. This finding revealed a striking difference in the posttranslational regulation of DNA packaging in Eukarya vs. the Archaea. This study illustrates a significant evolutionary step for the MS tools available for characterization of WT proteins from complex proteomes without proteolysis.
为了更全面地表征DNA预测的蛋白质（包括其翻译后修饰），本文提出了一种“自上而下”的方法，即使用高分辨率串联质谱，将其应用于产甲烷菌的假设驱动模式和发现模式，后者依赖于完整蛋白质的新自动化基准。使用二维蛋白质分级分离方法从詹氏甲烷球菌（约1800个基因）的核糖体和全细胞裂解物中分离蛋白质，通过在定制的四极杆/傅里叶变换混合质谱仪中对完整蛋白质离子进行自动碎片化，鉴定并表征了72个基因产物，序列覆盖率达100%。发现了三个错误的起始位点和两个修饰，其中各有一个是针对MJ0556确定的，MJ0556是一种20 kDa的蛋白质，在稳定期细胞中约50%占有率处存在未知甲基化。该分离方法与四极杆/傅里叶变换混合质谱仪相结合，能够有针对性地、高效地比较詹氏甲烷球菌、嗜乙酰甲烷八叠球菌（最大的古菌基因组，5.8 Mb）和酵母的组蛋白。这一发现揭示了真核生物与古菌在DNA包装的翻译后调控方面存在显著差异。这项研究说明了用于表征来自复杂蛋白质组的野生型蛋白质而无需蛋白水解的质谱工具的一个重要进化步骤。
