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Table 3. Technology Development Roadmap for Complex Identification and Characterization (acronyms)

Technology Objectives

Research, Design and Development

Demonstration: Pilots and Modular Deployment

Integration and Production Deployment

Facility Outputs

Develop technologies for complex identification and characterization

Analysis by mass spectrometry (MS):

  • Identification and quantification of both digested peptides and intact proteins

Data processing:

  • Data interpretation
  • Data archiving

Develop for mass spectrometry:

  • High-throughput complex analysis by MS
  • Improved data analysis
  • Improved methods for quantitation and determination of complex stoichiometry
  • Improved MS detection limits and dynamic range
  • Identification of protein complex modifications via top-down MS
  • Combined isolation and identification approaches
  • Improved online separations
  • Integrated, high-sensitivity analytical tools, eventually for single cells
  • Improved cleavage and digestion approaches
  • Improved ionization for broad classes of proteins
  • Microfluidic-based assays

Evaluate commercial hardware, software, and instrumentation

Pilot scale:

  • Optimization of protocols with regard to throughput, reproducibility, costs
  • Improved MS data-analysis tools
  • Database development and query tools

Assays:

  • Integrated "lab on a chip"
  • Probe-based affinity
  • Binding affinity
  • Automated neutron, cryoEM, and X-ray small-angle scattering
  • New technologies evaluated and incorporated
  • MS labeling for identification of contact interfaces

Establish high-throughput, automated pipelines:

  • Scale up via multiple parallel production lines

Refine QA/QA protocols

Automate data acquisition and data analyses

Evaluate and incorporate new technologies

Capability for high-throughput protein complex analysis by MS

Highly validated data of identified protein complexes

Confirmatory analyses of protein complexes via biophysical techniques

New tools for complex-identification analysis

Databases for complex identification and characterization

Examples:

  • Binding affinity
  • Interaction interfaces

Biophysical Characterization

Structural characterization

Binding affinities

Others

Establish structural and functional assays:

  • EM, SANS, SAXS, NMR
  • Approaches to identify contact faces
  • High-throughput binding affinity assay

To develop and incorporate the necessary technologies and methods into a high-throughput production environment, a phased process will be followed as described in this roadmap. The process includes research, design, and development; modular and pilot-scale deployment; and final integration and scaleup into operational procedures.