Biophysical Techniques in Proteomics and Protein Science
Dr. Rob McKenna
Course Objectives Course Outline Evaluations
The Omics Revolution
The Genomics Era
The en masse sequencing and characterizati
2-D Gel Analysis
ImageMaster & Melanie
Argonne National Labs Protein Mapping
Danish Centre for Human Genetics
What is Electrophoresis?
The migration of charged molecules in
solution in response to an electric field
Proteins, at a pH other than their pI,
carry a net charge
Rate is proportional to:
Processes which allow the resolution of solute
mixtures by selective fixation and liberation on a
solid support with the aid of directional fluid flow
Mikhail Tswett, 1901-1906
Separation of plant pigm
Tandem Analysis: LC -MS
Bells & Whistles, For What?
Simplicity Sensitivity Selectivity
LC-MS has a practical peptide maximum
Too many proteins/peptides swamps the analyzer At best, mixtures of 400-500 peptides are workable
2-D Gel Mass Spectrometry
Pick a Spot, Any Spot
2-D gel analysis
generates spots of
Visualize by stain
Pick by hand
Robotic spot picking
True Omics technology
High-throughput, en masse
Can be user directed or
Who Cares About MW?
LC-MS of Whole Protein?
LC will deconvolute your protein mixture
Separated proteins are then sent to MS
Highly accurate, high-tech SDS-PAGE?
Similar concerns as with 2D Gel-MS
Solution: Tryptic Peptides
LC III: HPLC
What is HPLC?
Originally referred to as High-Pressure
Now more commonly called High
Performance Liquid Chromatography
The instrument controlled version of LC,
utilizing very small particles, small column
MS II: Sample Ionization
Matrix-Assisted Laser Desorption
Fast Atom Bombardment
Electron Ionization (EI)
Hard ionization high degre
Multi-Dimentional Gel Electrophoresis
Whats Wrong With Just 1-D?
Co-migration of Protein
How many proteins in a cell weigh 50 kDa? How many proteins in a cell have a pI=6.4?
Picking Your Dimensions
2-D Separation based on similar properties will generat