Determination of IsoelectricPoint (pI) By Whole-Column

Detection cIEF

Tiemin Huang and Jiaqi Wu

CONVERGENTBIOSCIENCE

Determination ofIsoelectric Point (pI)

by Whole ColumnDetection cIEF

Definitionq The pH at which the net charge of an amphoteric compound is zero.q Determined by the dissociation of all ionizable functionalities of the amphoteric compound.q A physico-chemical parameter associated with every amphoteric compound.q Affected by parameters such as temperature, media compositions (dielectric constant), and ionic

strength (which influence the dissociation of ionizable functionality).

Conclusionq Thus, there is no such thing as an absolute pI value for a given amphoteric compound.

The pI determination method and conditions must be cited when stating a pI value.

Principle of Isoelectric Focusing (IEF)

When an amphoteric compound is placed ina medium with a pH gradient and subjectedto an electric field, it will move towards theelectrode with the opposite charge. As itmigrates, its net charge and mobility willdecrease and it will slow down. Eventually,the amphoteric compound will arrive at thepoint in the pH gradient where the pH isequal to its pI. Here it will be uncharged andstop migrating.

At this time, if the amphoteric compoundshould happen to diffuse to a region outsidethis pH (in the pH gradient medium), it willpick up a charge and hence move back tothe position where it is neutral. In this wayamphoteric compounds are condensed, orfocused, into sharp stationary bands

IEF process in an 100 mm ID- 50 mm longcapillary

Sample: 4 human hemoglobin variants0.5 min

1 min

1.5 min

2 min

2.5 min

3.5 min

4 min

5 min

6 min

Uniqueness of IEF

q Highest resolution of all the charge based separation techniques.*q Steady-state separation technique.*q Separation is relatively independent of sample load and the way the sample is introduced*

* Svensson H. Acta Chem. Scan. 1961, 15, 325-341.

Righetti P.G. Isoelectric focusing: theory, methodology and application; Elsevier Biomedical

Press: Amsterdam, 1983

pH Gradient Used in IEF

Natural pH gradient IEF

q Carrier ampholytes are hundreds or even thousands of amphoteric compounds specially synthesized tohave an even distribution of isoelectric points across a given pH ranges.

q The pH gradient forms naturally by carrier ampholytes under an electric field.q Can be conducted in gel format and capillary format (CIEF).q Resolving power about 0.02 pH unit.

Immobilized pH gradient IEF

q Gradient is formed by immobilized pH gradient gelq Highest resolving power about 0.001 pH unit .

Slab Gel IEF

q Commonly conducted on ready made or selfmaking agarose or polyacrylamide gel

q All steps in a gel IEF analysis are performedmanually§ Prepare slab gel (if pre-cast gel is used, this

step is not necessary)§ Load samples on the gel§ Fix the gel after electrophoresis§ Stain the proteins bands separated on the

gel§ Rinse the gel§ Analysis of protein band on the gel using an

imaging systemq Suitable for qualitative analysis, and less suitable

for quantitative analysis.q At present, the most popular IEF format although

it is labor intensive and time consuming (overhours)

Example of slab gel IEF Sample : Protein X

26917-70

Cathode

Anode

1A 2A 3 4 5 6 7 8

pI markersp

H g

rad

ien

t

Capillary Isoelectric Focusing (cIEF)

q At present, conducted with natural pH gradient IEF (carrier ampholytes-IEF) onlyq Automationq On-line detection and quantitationq Fast separation and detection (as short as 5 minutes )

pI marker 5.3

pI marker 7.4

Example of cIEFSample: Protein XpI values and area % are labeled in the e-gram

pI Determination

Standard Methods

• Computation from all ionizable functionalities of the amphoteric compound. That is basic andacidic amino acids present in the protein

• Titration

• cIEFa Measurement of pH after IEFb Calibrated from a mixture of pI markers

pI Determination

1. Algorithm Computation The negative charge (a ) carried by the acidic group of the ampholyte at a given pH can be expressed as

The positive charge carried by the basic group (a ) of the ampholyte at a given pH can be expressed as

Ê

The overall charge (Z) of the ampholytes is

Ê

The pI of the ampholyte (the pH where the net charge is zero) can be determined once all the pKa of the ampholytesare known.

ÊLimitations•Selecting different pK values gives different pI values (i.e., pI of peptide WDDD determined by (1) is 3.38, and it is calculated

to be 2.82 from the link (2).•The assumption that the ionization of ionizable groups is independent of the others is rarely true.•Modification of proteins is not taken in to account.•The actual folding pattern of the protein is not taken into account(1) Electrophoresis 2000, 21, 603-610(2) http://www.embl-heidelberg.de/cgi/pi-wrapper.pl

110

1)( +

= −−

pHpKa

110

1)( 2 +

= −+

pKpH

��=

−

=

+ −=n

ii

m

jjZ

11

pI Determination

2. Titration

A solution of the amphoteric compound ofinterest is prepared. The zeta potential over agiven pH range is recorded during titration.From the zeta potential (charge) versus pHcurve, the isoelectric point (pI) is the pH value ofthe point that the curve intercepts zero charge.The figure at the right illustrates the titrationcurve of peptide WDDD.

Limitation§ The isoionic point determined is influenced

by the buffer ionic strength*. Therefore, thepI based on titration may not be accurate

* Tanford C. Adv. Protein Chem. 1962,7, 69-165.

Velick S.F. J. Phys. Colloid Chem. 1949, 53, 135-149

-5

-4

-3

-2

-1

0

1

2

2 3 4 5 6 7 8 9

pH

Charg

e

pI Determination

3. cIEF

a. Measurement of pH after IEF

§ Isoelectric point for a protein isdetermined by measuring the pH of theprotein band or spot on an isoelectricfocusing gel

§ The pI determined is accurate whenexperimental temperature is controlled.

ÊLimitations§ Can only be used for gel IEF, and not

practical for CIEF at present.§ The influence of carbon dioxide to the gel

system has to be controlled

Measurement of pH values on IEF gel

Anodic

Cathodic

pH meterWith microprobe

pI Determination

b. Calibrated by a Mixture of pI Markers

§ The pI is determined by using a series ofcalibrated mixture of pI markers

§ Performed on slab gel IEF and cIEF.

Limitations§ Assume the pH gradient is linear.§ Assume the given value of pI markers is

correct

pI markers bands

Gel IEF: pI range of the sample is in 7.5 Ð 8.4

Unknown samples

MarkerMarker

Marker

Unknown sample

7.67.7

7.9

cIEF

Instrument Usedfor cIEF Method

DialysisHollow Fiber

IEF Column Inlet CapillaryOutlet

H+ OH-

+ -

Detector:Camera in UV

Light Beam at 280 nm

Focused Zones

Sampleinjection

iCE280 Analyzer

pI DeterminationUsing cIEF Ð

Ideal Conditions

q pI markers with accurate pI values

q Linear pH gradient created by used carrier ampholytes(usually a single carrier ampholyte is used)

Ideal Conditions ÐDetermining

Markers’ pI Values

q 15 synthesized peptides are used as the pImarkers (Electrophoresis, 21, 603(2000))

q pI values of the markers are measured bymeasuring pH along IEF gel after IEF of themarkers

Measurement of pH values on IEF gel

Anodic

Cathodic

pH meterWith microprobe

2

3

4

5

6

7

8

9

10

11

0 500 1000 1500 2000

Peak position (pixel)

pI valu

e

Ideal Conditions ÐDetermining pH

Linearity forCarrier Ampholytes

3

4

5

6

7

8

9

10

0 500 1000 1500 2000

Peak position (pixel)

pI v

alu

e

Ampholine 3.5-9.5

3

4

5

6

7

8

9

10

0 500 1000 1500 2000

Peak position (pixel)

pI

va

lue

Biolyte 3-10

Servalyt 2-11

3

4

5

6

7

8

9

10

0 500 1000 1500 2000

Peak position (pixel)

pI v

alue

Pharmalyte 3-10

Ideal Conditions ÐDetermining pH

Linearity forCarrier Ampholytes

3

4

5

6

7

8

9

10

0 500 1000 1500 2000

Peak position (pixel)

pI v

alue

r2=0.997r2=0.999

r2=0.998

8

8.5

9

9.5

10

10.5

500 1000 1500 2000

Peak position (pixel)

pI v

alue r2=0.999

5

5.5

6

6.5

7

7.5

8

300 800 1300 1800

Peak position (pixel)

pI valu

e

r2=0.993

2.5

3

3.5

4

4.5

5

0 500 1000

Peak position (pixel)

pI v

alu

e r2=0.995

Pharmalyte 3-10

Pharmalyte 2.5-5

Pharmalyte 8-10.5

Pharmalyte 5-8

Accuracy in pIDetermination

under Ideal Conditions

Ifu The pH gradient linear correlation coefficient r2 > 0.99,u and the distance between the two used pI markers < 2 pH units

Then,The accuracy in pI determination is estimated to be< ±0.15 pH units*

*σ=(Syy*(1-r2))1/2 =(2*(1-0.99))1/2 =0.14

Syy=Σ(pIi-pI)2 pI is the expected pI value of this marker if the pH gradient is linear

pI Determinationunder Ideal

Conditions Ð Example

Sample: Mab1

§ The pI value of this Mab is known to be in pH 5.5 Ð 7region

§ The pH gradient of the used carrier ampholyte(Pharmalyte 3-10) has good linearity around pH 5.3Ð 7.3 region (r2=0.999)

3

4

5

6

7

8

9

10

0 500 1000 1500 200

pI

r2=0.999in pI 5.3 Ð 7.3 range

pI Determination underIdeal Conditions ÐExample (Cont’d)

Sample: Mab1

§ Run the Mab with two pI markers in the linear pHregion

§ To achieve optimal accuracy, select the two pImarker peaks that bracket the unknown samplepeaks

§ pI value of the Mab is calculated using the pI valuesof the two pI markers*

5.31

7.27

Mab1

The pI of the major peak of this Mabis determined to be 6.06

*F=(position of pI7.27 marker - position of pI5.31 marker)/(7.27-5.31)

Sample’s pI=5.31+F*(sample position-position of pI5.31 marker)

pI DeterminationUsing cIEF Ð

Compared to Calculation

q pI values measured by cIEF is based on surface charges of a protein

q In the calculation, the actual folding pattern of the protein is not taken into account

q We have found that the pI values obtained from cIEF under denatured conditions(unfolded protein) are closer to that of calculated ones

pI DeterminationUsing cIEF Ð Compared

to Slab Gel IEF

q Different pI markers and carrier ampholytes used in cIEF (usually small moleculemarkers) and gel IEF (usually proteins markers) may create differences

q Protein markers usually have multiple peaks

q In slab gel IEF, matrix effect on pH gradient is usually not compensated unlessinternal pI markers are applied in samples

pI Determination in CarrierAmpholyte Mixture

(Non-Ideal Condition)

The pH gradient created by carrier ampholyte mixture is usually non linear:

q Bigger error may involve in pI determination§ The accuracy is difficult to estimate

pI Determination inCarrier AmpholyteMixture - Example

0 500 1000 1500 2000

3

4

5

6

7

8

9

0 500 1000 1500 2000

Peak position (pixel)

pI

va

lue

pI markers in 2% pH2.5-5 Pharmalyte and 2% pH3-10 Pharmalyte

pI Determination in CarrierAmpholyte Mixture Ð

Example (Cont’d)

§ Two pI markers are required in the sample to compensate the matrix effect of the sample§ The two markers’ peaks should bracket the sample peaks§ The two markers are also included in the marker standard e-gram

0 500 1000 1500 2000

0 500 1000 1500 2000

pI markers in 2% pH2.5-5 Pharmalyte and 2% pH3-10 Pharmalyte

Sample A in 2% pH2.5-5 Pharmalyte and 2% pH3-10 Pharmalyte

pI Determination in CarrierAmpholyte Mixture Ð

Example (Cont’d)

0 500 1000 1500

0 500 1000 1500 2000

u The sample e-gram is stretched and aligned to the marker standard e-gram (acts as a ruler)u The pI values of sample peaks are estimated by assuming pH gradient linearity between any two pI

markers in the marker standard e-gram

pI markers

Sample

Sample Peak Identificationin cIEF

q Identify the same peaks in different samples

q This identification does not require accurate pI determination§ “Measured pI values” of the sample peaks under the same sample running

condition may be used in the identification in the same way as the “relativeretention time” in chromatography

q The identification can be performed regardless of pH gradient linearity (single carrier ampholyteand carrier ampholyte mixture can be used)

q Only two pI markers are needed to perform the identification

Sample Peak Identification ÐNeed for Internal pI Markers

q Different matrices§ Peak may shift in some matrices

q Different sample concentration§ Different salt concentration due to different dilution factor§ Salts squeeze pH gradient created by carrier ampholytes

q Samples from different sources§ In different matrices

Two internal pI markers can compensate for these effects§ pI marker peaks should bracket the sample peaks

Sample Peak Identificationin Single Carrier

Ampholytes Ð Procedure

q Run all samples with the same two pI markers§ pI marker peaks should bracket the sample peaks

q Measued pI values of all sample peaks are calculated using the pI values ofthe two pI markers by assuming the linearity between the two pI marker peaks§ F=(pI value difference between the two pI markers)/(peak position

of second pI marker Ð peak position of first pI marker)§ Sample’s measured pI=first pI marker’s pI value+F×(sample

peak position-peak position of first pI marker)§ First pI marker is the marker with lower pI value

q The measured pI values are used to identify the sample peaks

Sample peaks are identifiedby their measured pI values

Sample Peak Identificationin Single Carrier

AmpholyteÐExample

pI marker 5.3 pI marker 6.67.5 mM PBS

15 mM PBS

25 mM PBS

Peak pattern is squeezed by salts

Peak position differences > 0.03 pI (measured value)can be identified

In pH 4 Ð 7 Servalyt

Position

Measured pI

5.96 6.

015.

89 6.11

6.21

5.97 6.

025.

91 6.12

6.22

5.91

5.97

6.02

6.12

6.22

Sample Peak Identificationin CarrierAmpholyteMixture Ð Procedure

q Run all samples with the same two pI markers§ pI marker peaks should bracket the sample peaks§ Proportion of each carrier ampholyte in the carrier ampholyte mixture should

be strictly controlled in order to obtain good precision» Pre-mixed carrier ampholytes stock solution is a good idea

q Overlay e-grams of all samples by aligning the two pI marker peaksq The sample peaks are identified by their positions in the overlaid e-grams

Sample Peak Identificationin Carrier Ampholyte

Mixture Ð Example

pI marker 3.78

pI marker 9.50

pI marker 3.78

pI marker 9.50

Sample A in different salt concentrations

Sample in 2% pH2.5-5 Pharmalyte and 2% pH3-10 Pharmalyte

Sample Peak Identificationin Carrier Ampholyte

Mixture Ð Example (Cont’)

pI marker 3.78

pI marker 9.50

pI marker 3.78

pI marker 9.50

Sample in 2% pH2.5-5 Pharmalyte and 2% pH3-10 Pharmalyte

Overlay and align the two e-grams by the two pI marker peaks

Sample peaks are identified by their positions in the e-grams