Proteins separation and analysis Proteins separation and analysis using Fast Protein Liquid using Fast Protein Liquid

ChromatographyChromatography

Ayelet David, Ph.D

Dept. of Clinical Pharmacology

Fast Protein Liquid Chromatography (FPLC)

Column chromatography used to separate or purify proteins from complex mixtures based on size, charge distribution, hydrophobicity of biorecognition (affinity chromatography).

Typical columns used for protein purification, include:

• Gel Filtration Chromatography — separate proteins according to their size. Also termed as “size exclusion chromatography” (SEC).• Ion exchange chromatography — separate proteins based on surface-charges.• Reversed phase or hydrophobic interaction — separates based on hydrophobicity.• Affinity chromatography — separates based on ligand affinity, such as a His-tagged protein would use a nickel column.

Gel Filtration Chromatography - Gel Filtration Chromatography - Principle

Molecules elute in order of size.The largest molecules come

first; other molecules leave the column in decreasing order of size; the smallest ones come last.

Stationary phase

pore

Crossed-linkedmatrix

Stationary phase: porous, cross-linked beads(dextran, agarose, polyacrylamide)

degree of cross-linking determines diameter of pores and fractionation range of biomolecules of different size

proteins do not attach to column

• Scanning electron micrograph of an agarose gel, Magnification x 50,000• The chromatographic medium is a gel. • The gel is in the form of beads.

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Gel structure

AGAROSE

A good gel for gel filtration contains about

95% water

Ref: Anders S. Medin, PhD Thesis, Uppsala University 1995

Biomolecules are separated according to size Biomolecules are separated according to size (hydrodynamic volume)(hydrodynamic volume)

A gel beads that encloses an internal solvent space. Smaller molecules (red) can freely enter the internal solvent space of the gel bead, whereas larger molecules (blue) are too large to penetrate the gel pores.

Separation according to sizeSeparation according to size

9

Void volume Vo

Volume of the gel matrix Vg

Pore volume Vi

Vo= Void volume

Ve = Elution volume within the separation

range of the gel

Vi = Inner pore volume = Vc - Vg - Vo

Vc = Total (geometric) volume of the column

Vt = Total volume of the column

2 31

Vo

Ve

Vt

Vc

Terms and explanations

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The void volume Vo

Vo Ve

Volume

Con

cent

rati

on

Elution volume for very large molecules, Vo

All molecules larger than the largest pores will elute together at the void volume. Ve = Vo

For most gel filtration columns, the void volume represents 30 to 40 % of the total column volume Vc.

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Vt and Vc

Volume

Con

cent

rati

on

VtVc

Vo Ve

Elution volume for very small molecules, Vt

Geometric volume of the gel bed, Vc

The volume in which a small molecule elutes from the column is V t

Vt = Vo + Vi

Vt is slightly smaller than Vc.

12

The distribution coefficient, Kd

i

oe

goc

oe

idoe

V

VV

VVV

VVd

VKVV

K

Kd is difficult to get because Vi is difficult to measure

Kd = the fraction of the stationary phase which is available to a given solute. It depends only on the gel and the size of the solute.

13

The coefficient Kav

ot

oe

iavoe

VV

VVav

VKVV

K

Kav is easy to get and it is more useful in practiceKav is not a true partition coefficient

Since the Vi is difficult to measure, it is usual to substitute the term (Vt-Vo) for Vi in the partition equation and call the result Kav instead of Kd.

14

Kav for very large and very small molecules

0

oVat eluting molecules largeFor very

ot

oo

ot

oe

e

VV

VV

VV

VVav

o

K

VV

1

tVat eluting molecules smallFor very

oe

oe

ot

oe

e

VV

VV

VV

VVav

t

K

VV

15

Kav should always be in the range 0 to 1

Vt

Ve

Volume

Con

cent

rati

on

Elution volume when Kav = 1

Elution with Kav > 1Adsorption has occurred

Some typical applications for gel filtration

1. Group separation: Desalting, Buffer exchange, Removing reagents

2. Purification of proteins and peptides: complex samples, monomer/dimer

3. Estimation size & size homogeneity

Sephadex G-25 is widely used for these applications. Its separation range makes it suitable for group separations work, such as the removal of salt contaminants from molecules larger than about 5,000.

Application 1Application 1 - - Desalting and buffer exchange

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Desalting proteins

Desalting in a simple column

aa

2Elution volume (ml)

0 4 6 8 10 12

Albumin NaCl

Column:

Sample:

Buf fer:

PD-10

HSA, 25 mg

NaCl 0.5M

(Sephadex G-25)

Application 2Application 2 - - Purifications of proteins

A gel filtration column with two different size molecules applied. The larger molecules exit the column first

Separating dimer and oligomers from monomer

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Separating dimer and oligomers from monomer

Column: Superdex 75 HR 10/30 Sample: A special preparation ofrhGH in distilled water

0.025

0.05

Oligomer

Monomer

ime (min)

Dimer

T10 20VO VC

280 nmA

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Selectivity curves

Plotting Kav versus the logarithm of molecular mass yields the selectivity curve of the matrix.

The steeper this curve, the greater the difference in elution volume for two molecules of different sizes.

Kav

log Mr

Kav

log Mr

Kav log Mr

There is a sigmoidal relationship between Kav and the logarithms of the molecular masses for molecules of similar shape. Over a considerable range, a linear relationship exists.

Application 3Application 3 - - Determination of molecular weight

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Constructing a selectivity curve

Kav 1

0log (Mr)

1. Run standards and determine the elution volume for each.

2. Calculate Kav values.

3. Plot log (Mr) for each standard against the calculated Kav.

• A selectivity curve is fairly linear between Kav values of 0.1 and 0.7

• The molecular weight range which lies between these values is defined as the useful fractionation range of the medium.

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Exclusion limit

Kav 1

0log (Mr)

Exclusion limitAll molecules bigger than this elute in the void volume

The exclusion limit is the molecular weight of the smallest molecule which cannot enter the pores of the matrix. It is an extrapolated value defined by convention.

Gel matrix has optimum ~ linear range

AKTAAKTAFPLC SystemFPLC System

All instrument settings and functions are under the direct control of UNICORN, a real-time control system.

Syringe pump producing accurate, reproducible, pulse free flow rate and a precise gradient formation

On line monitor offering the possibility to measure UV conductivity, and pH.

Fraction collector

A seven port motorized valve, used for sample application

Injection valve INV-907

A single chamber mixer, powered and controlled from Pump P-920

Mixer M-925Column

Column is easily placed on the outside of the system

UV detector

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Estimating molecular size

Measure elution positionMeasure elution position

Calculate molecular sizeCalculate molecular size

All data from a run, including running conditions, method and start protocol, and a complete log of every event during the run, are automatically stored in a single file.

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Resolution depends on efficiency and selectivity

High efficiency

Low efficiency

High selectivity

Low selectivity

Efficiency is a measure of peak width

Selectivity is a measure of peak separation

Efficiency is governed by the separation medium, how well the column has been packed, and the running conditions.

Selectivity is governed almost entirely by the separation medium itself, so choosing the right medium is essential to obtaining the right selectivity for a given separation

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Resolution Rs

Rs = 4

Rs = 0.6

Rs = 1

RV VW W

sr r

2 1

1 2

2

Resolution = Peak separationAv. peak width