Targeted analysis and discovery of posttranslational modifications in proteins from methanogenic archaea by top-down MS

Andrew J. Forbes, Steven M. Patrie, Gregory K. Taylor, Yong Bin Kim, Lihua Jiang, Neil L. Kelleher

Research output: Contribution to journalArticle

78 Scopus citations

Abstract

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 (≈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 ≈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.

Original languageEnglish (US)
Pages (from-to)2678-2683
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number9
DOIs
Publication statusPublished - Mar 2 2004

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ASJC Scopus subject areas

  • Genetics
  • General

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