Physical Methods to Characterize Proteins

Molecular weight

Physical properties of key interest

Oligomerization state

Structure

Interactors

Transport Processes

1. Transport in an electric field (electrophoresis)

2. Transport in a gravitational field (centrifugation)

3. Transport by partitioning between mobile and stationary phases (gel exclusion chromatography)

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.

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.

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

Leading anion: Cl-

Trailing ion: glycine

Changes in electrophoretic mobility in the focusing zone

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

Basic mass spectrometer design

Detection methods

Time of Flight (TOF)- Accuracy to 0.1%

Quadrupole mass analyzers- Accuracy to 0.01%

FT ion cyclotron- Accuracy to 0.001%

Ionization methods

Matrix-assisted Laser desorption/ionization (MALDI)

Electrospray ionization (ESI)

Mass determination of intact proteins

Tandem mass spectrometry

Tandem mass spectrometry analysis by collision-induced dissociation (CID)

Types of MS Data

SILAC-Stable Isotope Labeling by Amino Acids