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Module 5: A Detailed Discussion of Top-Down Proteomics Methodologies

FACULTY

Scot R. Weinberger, President, CEO and Founder of GenNext Technologies™ Inc.

Dr. Tracy M. Andacht, Director and co-Founder of the Proteomics Resource Facility at the University of Georgia

David Garfin, PhD, President of The American Electrophoresis Society

Dr. Ron Orlando, Assistant Professor Complex Carbohydrate Research Center and Departments of Biochemistry & Molecular Biology and Chemistry University of Georgia

This module is comprised of five different areas of focus: 2-D Gels and Differential Gel Electrophoresis; Virtual 2-D Gel Analysis; SELDI; Top-Down LC-MS; and Top-Down FTICRMS. At the completion of this module group, students will be capable of effectively integrating experiments and results among all top-down platforms as well as correlating scientific results with bottom-up proteomic approaches. A balanced emphasis upon protein identification as well as phenotypic patterns will be struck.

2-D GELS AND DIFFERENTIAL GEL ELECTROPHORESIS: This module will familiarize students with 2DE as a way to prepare and analyze protein expression maps. Students will be instructed in sample-preparation methods and trained in all phases of 2DE including running gels, staining proteins in gels, image acquisition, and image analysis. Students will be introduced to the various staining and labeling techniques, and the advantages and disadvantages of each will be discussed. Students will become familiar with the processing of gel plugs for MALDI MS and peptide mass fingerprinting for protein identification, as well as issues that limit its success. Using Differential Gel Electrophoresis (DIGE) experimental data as an example and experimental design considerations will be discussed to allow students an opportunity to become familiar with software aided gel analysis. By the end of the course, students are expected to have a firm foundation in 2DE and capable of running hypothesis or phenomenological driven gel experiments from protein extraction to data analysis.
(Tracy Andacht and David Garfin)

VIRTUAL 2-D GEL ELECTROPHORESIS: This module serves to provide the students with an overview and historical perspective of virtual 2-D gel electrophoresis. We will examine the pioneering direct combination of IEF with mass spectrometry that served as a driving impetus for the creation of today's chromato-focusing-RP MS platforms. Students are expected to understand the inherent difficulties in directly hyphenating gels with MS, while simultaneously developing an appreciation of the resultant analytical power of this approach.
(Scot Weinberger)

PROTEIN BIOCHIPS / PROTEIN ARRAYS: Protein Biochips or Protein Arrays are fundamentally an arrangement of multiple miniaturized probe sites in a single, compact analytical surface. Arrays can be thought of as two-dimensional structures (classic planar surfaces or modified microtiter plates) as well as three-dimensional devices (interaction beads, nanoparticles, or microtubes). Detection of adsorbed proteins is accomplished using a variety of direct or indirect schemes such as Laser Induced Fluorescence, Chemiluminescence, Surface Plasmon Resonance Optical Spectroscopy, Grating Coupled Waveguide Refractive Index Spectroscopy, Interferometry, Imaging Ellipsometry, Atomic Force Microscopy, Ultra-sonic Acoustic Detection, Electrical Conductivity Detection, and Mass Spectrometric Detection. Protein Arrays have been progressively used in translational proteomic studies to discover biomarkers, understand protein interactions and signaling pathways, as well as to elucidate protein function.

This learning module will provide basic understanding of the principles of Protein Array analysis, including detailed discussion of array surface chemistries, protein content (bait), operational schemes, and their practical uses. Students are expected to gain an understanding of the role that protein biochip technology and tools can play towards enabling top-down proteomic studies. Students are further expected to be capable of designing and performing de novo and directed biomarker discovery experiments. Additionally, students are expected to understand how to combine the results from top-down protein array studies with bottom-up proteomics methodologies as well as gene array expression analysis.
(Scot Weinberger)

TOP-DOWN LC-MS: This module describes the history and advancement of top-down LC-MS methodologies. At the completion of this module, students are expected to understand the capabilities of Top-Down LC-MS analysis, appreciate the difference between chromato-focusing and iso-electric focusing, and be capable of designing experiments using these technologies. Furthermore, students will be able to design experiments that effectively leverage the complementary nature of Top-Down LC-MS analysis with that of gel, virtual gel, and SELDI analysis.
(Scot Weinberger)

TOP-DOWN FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY (FTICR MS): This learning module is intended to provide students with an enabling and basic understanding of the principles of FT-MS analysis. A technical overview is provided, including detailed discussion of FT-MS instrumentation, ECD, and IRMPD. Particular emphasis is given towards the design and execution of successful top-down translational proteomic studies. By participating in this module, students are expected to understand the role that FT-MS technology can play towards enabling top-down proteomic studies. Additionally, students are expected to understand how to combine the results from top-down FT-MS studies with the other top down and bottom up proteomics methodologies as well as gene array expression analysis.
(Ron Orlando)

GenNext's 2007 In Person Translational Courses have been completed. GenNext would like to thank its Education Partners and excellent instructors for another highly received Translational Research Program.

Information regarding GenNext's 2008 program will become available shortly. Sign-up to find out about upcoming GenNext courses.

For more information about this course, please contact GenNext Technologies at inform@gennexttech.com or call 650-563-9577.