Transcript Physical Characterization of Proteins

Physical Methods to
Characterize Proteins
Physical properties of key interest
Molecular weight
Oligomerization state
Structure
Interactors
Transport Processes
1. Transport in an electric field (electrophoresis)
Requires a matrix (either agarose or polyacrylamide) to
minimize convective effects due to heating and Brownian
motion. Mobility is also affected by partioning with the
stationary phase (matrix). Provides relative molecular
weights determined by comparison to standards.
2. Transport in a gravitational field (centrifugation)
Requires a centrifuge to generate the large gravitational
forces required for protein transport. Provides an
absolute molecular weight and informs on the
oligomerization state for stable complexes.
3. Transport by partitioning between mobile and
stationary phases (gel exclusion chromatography)
Requires a suitably-sized partitioning matrix for the solid
phase. Provides relative molecular weights determined by
comparison to standards.
Gel Electrophoresis
u = A(q/f)
Electrophoretic mobility (u) is proportional to its net charge (q), specifically the
ratio of its net surface charge to accessible surface area, and inversely
proportional to its frictional coefficient (f), a function of solvent viscosity and
protein geometry. The constant of proportionality (A) is unique to each protein.
Mobility can be measured to the cathode
or anode depending on protein charge
and pH.
Free radicals are provided by ammonium persulfate.
TEMED (tetramethylenediamine) is included to stabilize
the free radicals resulting from decomposition of the
ammonium persulfate. Ratio of acrylamide to
bisacrylamide is held constant. Pore size is expressed
as percent (w/v) acrylamide and designated %T.
Disc (Discontinuous) Gel Electrophoresis
Changes in electrophoretic mobility in
the focusing zone
Leading anion: ClTrailing ion: glycine
SDS PAGE
SDS: Sodium Dodecyl Sulfate (Lauryl sulfate)
SDS binds protein with a constant ratio of 1.4
gm SDS/gm protein.
Calibration plot for a series of molecular
weight standards vs. %T
Gel Filtration/Size Exclusion Chromatography
Comparison of size resolution by gel
filtration (left) and PAGE (right)
Band spreading as a function of
elution position
Calibration plots for molecular weight
standards resolved on different media
Mass Spectrometry
Ionization methods
Matrix-assisted Laser desorption/ionization (MALDI)
Basic mass spectrometer design
Electrospray ionization (ESI)
Detection methods
Time of Flight (TOF)- Accuracy to 0.1%
Quadrupole mass analyzers- Accuracy to 0.01%
FT ion cyclotron- Accuracy to 0.001%
Types of MS Data
Mass determination of intact proteins
Tandem mass spectrometry analysis by collisioninduced dissociation (CID)
Tandem mass spectrometry
SILAC-Stable Isotope Labeling by Amino Acids