| This study describes the development and application of a “Top Down” proteomics platform, where intact proteins were efficiently fractionated, identified and characterized. Using this “top down” approach, intact protein ions were directly fragmented inside an Electrospray (ESI) Fourier-Transform (FT) mass spectrometer, which allows efficient DNA-predicted protein identification and characterization. Such method enables effective detection of general (or even novel) post-translational modifications.; The platform we developed mainly involves three elements: In the “Front End”, we utilized an Acid-Labile Surfactant-based gel electrophoresis and Reversed-Phase Liquid Chromatography to efficiently fractionate whole cell lysates. In the middle, automated Q FTMS was employed to directly analyze intact protein samples. At the “Back End”, we developed and optimized several computer programs to facilitate reduction of FTMS data. The acquired MS and MS/MS data were then used to identify intact protein forms from a protein sequence database.; We applied this top down proteomics platform to study proteins from Methanococcus jannaschii and Saccharomyces cerevisiae. From the whole cell lysate of yeast, ∼120 ion species were successfully identified with 100% sequence coverage, and we also detected several post-translational modifications, including acetylations, phosphorylations, methylations, formylations and oxidations. |
