Proteomics

Leadership

Arthur Moseley, PhD
919-684-4456
arthur.moseley@duke.edu

URL: http://www.genome.duke.edu/cores/proteomics/

Resource Overview

The Duke Proteomics Core Facility for LC/MS analyses, as a shared resource, provides the Duke research community access to a comprehensive suite of state-of-the-art proteomic analyses, from the qualitative identification of simple protein mixtures (gel spots/bands) to the qualitative and quantitative analyses of complex mixtures (cell/ tissue lysates and biological fluids).

The capabilities of the resource were designed to support both basic sciences and clinical sciences, and the scale of the facility was designed for support of clinical studies, including biomarker discovery and biomarker verification.

This centralized Proteomics Resource makes expensive and sophisticated technologies accessible to all interested researchers, providing an infrastructure comprised of more than $5,000,000 of LC/MS/MS instrumentation, two dedicated servers for data processing, and a staff of seven proteomic experts who have more than 40 years of combined proteomic mass spectrometry experience.

This state-of-the-art proteomics toolkit coupled with the experience and skill of the staff insures that maximum value will be derived from the experiments utilizing the resource.

The experimental design of all proteomics experiments are created via collaborative discussions between senior staff members of the Proteomics Core Facility and the Investigators.

This collaborative creation of the experimental design has proven to be essential, as it insures that the facility staff have a clear understanding of the desired metrics of success for the biological/ clinical project, and the biological/ clinical collaborators are fully informed as to the special requirements of the preparation of samples quite different from a traditional protein chemistry sample preparation procedures.

Biostatisticians from the Institute for Genome Sciences & Policy are often included in the experimental design discussions, to insure proper powering of the experiment (number of biological and technical replicates).

Rigorous Quality Control samples and processes are followed to insure the highest quality results, providing both low False Positive Discovery Rates (< 1 percent) with high quantitative reproducibility (<20 percent relative standard deviation) for many thousands of peptides per sample.

All LC/MS/MS analyses are performed by full time staff members of the Proteomics Core Facility. Collaborators are trained by the resource personnel in the use of software for data mining and data interpretation, and their data is returned to them in a secure Web-based manner via the IGSPnet.

Capabilities

• Open (unbiased) qualitative and quantitative analyses using high resolution, accurate mass data for high confidence identifications and good quantitative reproducibility -- preferred tool for differential protein expression & biomarker discovery
• Targeted protein quantitation for high sensitivity, high specificity and excellent
  quantitative reproducibility -- preferred tool for protein expression verification
• Characterization of post-translational modifications, including phosphorylation
• Multidimensional characterization of gas-phase structures of peptides, intact
  proteins and protein complexes based on mass, size, shape, and charge

Equipment

• Qualitative and quantitative LC/MS/MS systems -- Four nanoscale capillary ultraperformance LC systems (Waters NanoAcquity); two multidimensional capillary nanoscale system (Waters NanoAcquity LC/LC); five high resolution, accurate mass, tandem mass spectrometers
• One hybrid quadrupole/time-of-flight systems (Waters Ultima)
• Three hybrid quadrupole/traveling wave ion-mobility/time-of-flight
  tandem mass spectrometers (Waters Synapt HDMS systems -- 
  one G1 and two G2 systems)
• One hybrid linear ion-trap/three dimensional ion-trap (Thermo LTQ-Orbitrap XL); one triple quadrupole tandem mass spectrometer (Waters Xevo)
• Informatics -- qualitative analyses
   - Data dependent acquisition database searching
• Dell Blade Cluster (six Blades, 48 processor equivalents)
• Matrix Science Mascot software (40 processor license)
  - Data independent database searching
• Supercomputer GPU workstation (448 GPU cores) (Waters IdentityE Software)
   - Data visualization software (Proteome Software Scaffold)
• Informatics -- quantitative analyses
   - Dell Server R900 (Two quad core processors, 16 processor cores, 64
    GB of RAM)
   - Rosetta Elucidator software (statistics, visualizations)
• Informatics -- data storage
  - 72 terabytes of enterprise quality Storage •Data mirrors for data security