Design of the first analytical platform for automated top down proteomics with quadrupole/Fourier transform ion cyclotron resonance mass spectrometry

Systems biology is directed at understanding the progression from a derived genome sequence to an expressed phenotype. The sequencing of genomic DNA creates the underlying infrastructure required for understanding biological questions and has led to an explosion in technology directed at high-throughput analysis. Mass spectrometry, coupled with improved separations technologies and genome predicted protein databases, has emerged as a dominant analytical technique for the determination of expressed gene products, allowing the identification of thousands of proteins from a single cell culture.; The greatest impact of these technologies is yet to be realized as they are moved from proof-of-principle to "real world" applications. One major limitation observed thus far is proteins are identified with poor coverage of the primary amino acid sequence. This constrains characterization of different protein forms with post-translational modifications (PTM's) that may be regulated during cellular growth. To overcome this, proteins may be viewed in their intact form (Top Down proteomics). Knowledge of the intact mass leads to immediate characterization of the entire amino acid backbone. A prominent analytical technique for intact protein analysis is Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS); which is a leader in reliable mass accuracy and resolution for a variety of biomolecules.; The enclosed writing details technology development leading to the first high-throughput proteome analysis of Methanosarcina acetivorans utilizing a custom FT mass spectrometer. It outlines a two dimensional separations platform that utilizes a 96 well plate nanospray robot to facilitate automated analysis on the mass spectrometer. This thesis also highlights instrument advancements required to improve throughput for observation of protein intact masses and methods used to obtain robust fragmentation. Included within the automation platform is software developed for efficiently managing intact protein and MS/MS data. Also, a novel fragmentation method has been developed to extend dynamic range for fragmentation of intact proteins as large as 60 kDa. The enclosed work is the foundation for the first fully automated platform for intact protein analysis by tandem MS. With the identification of hundreds of proteins from a single run, the high-throughput Top Down proteome analysis of intact proteins is now a reality.