Determination of Isoelectric Point (pi) By Whole-Column Detection cief Tiemin Huang and Jiaqi Wu CONVERGENT BIOSCIENCE
Determination of Isoelectric Point (pi) by Whole Column Detection cief Definition The ph at which the net charge of an amphoteric compound is zero. Determined by the dissociation of all ionizable functionalities of the amphoteric compound. A physico-chemical parameter associated with every amphoteric compound. Affected by parameters such as temperature, media compositions (dielectric constant), and ionic strength (which influence the dissociation of ionizable functionality). Conclusion 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 in a medium with a ph gradient and subjected to an electric field, it will move towards the electrode with the opposite charge. As it migrates, its net charge and mobility will decrease and it will slow down. Eventually, the amphoteric compound will arrive at the point in the ph gradient where the ph is equal to its pi. Here it will be uncharged and stop migrating. At this time, if the amphoteric compound should happen to diffuse to a region outside this ph (in the ph gradient medium), it will pick up a charge and hence move back to the position where it is neutral. In this way amphoteric compounds are condensed, or focused, into sharp stationary bands IEF process in an 100 mm ID- 50 mm long capillary Sample: 4 human hemoglobin variants 2 min 2.5 min 3.5 min 4 min 5 min 6 min 0.5 min 1 min 1.5 min
Uniqueness of IEF Highest resolution of all the charge based separation techniques.* Steady-state separation technique.* 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 Carrier ampholytes are hundreds or even thousands of amphoteric compounds specially synthesized to have an even distribution of isoelectric points across a given ph ranges. The ph gradient forms naturally by carrier ampholytes under an electric field. Can be conducted in gel format and capillary format (CIEF). Resolving power about 0.02 ph unit. Immobilized ph gradient IEF Gradient is formed by immobilized ph gradient gel Highest resolving power about 0.001 ph unit.
Slab Gel IEF Commonly conducted on ready made or self making agarose or polyacrylamide gel All steps in a gel IEF analysis are performed manually Prepare slab gel (if pre-cast gel is used, this step is not necessary) Example of slab gel IEF Sample : Protein X Cathode 26917-70 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 system ph gradient Anode 1A 2A 3 4 5 6 7 8 Suitable for qualitative analysis, and less suitable for quantitative analysis. At present, the most popular IEF format although it is labor intensive and time consuming (over hours) pi markers
Capillary Isoelectric Focusing (cief) At present, conducted with natural ph gradient IEF (carrier ampholytes-ief) only Automation On-line detection and quantitation Fast separation and detection (as short as 5 minutes ) pi marker 7.4 pi marker 5.3 Example of cief Sample: Protein X pi 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 and acidic amino acids present in the protein Titration cief a Measurement of ph after IEF b 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 1 = ( pka ph ) The positive charge carried by the basic 10 group (a + 1) of the ampholyte at a given ph can be expressed as The overall charge (Z) of the ampholytes 10 is 1 + = ( ph pk2 ) + Z = m j= 1 + j n i= 1 1 i The pi of the ampholyte (the ph where the net charge is zero) can be determined once all the pka of the ampholytes are known. Limitations 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
pi Determination 2. Titration A solution of the amphoteric compound of interest is prepared. The zeta potential over a given ph range is recorded during titration. From the zeta potential (charge) versus ph curve, the isoelectric point (pi) is the ph value of the point that the curve intercepts zero charge. The figure at the right illustrates the titration curve of peptide WDDD. Charge 2 1 ph 0 2 3 4 5 6 7 8 9-1 -2-3 -4-5 Limitation The isoionic point determined is influenced by the buffer ionic strength*. Therefore, the pi 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
pi Determination 3. cief a. Measurement of ph after IEF Measurement of ph values on IEF gel Anodic Isoelectric point for a protein is determined by measuring the ph of the protein band or spot on an isoelectric focusing gel The pi determined is accurate when experimental temperature is controlled. Limitations 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 Cathodic ph meter With micro probe
pi Determination b. Calibrated by a Mixture of pi Markers The pi is determined by using a series of calibrated mixture of pi markers Performed on slab gel IEF and cief. pi markers bands Unknown samples Limitations Assume the ph gradient is linear. Assume the given value of pi markers is correct Gel IEF: pi range of the sample is in 7.5 н 8.4 7.9 7.7 7.6 Marker Marker Marker Unknown sample cief
Instrument Used for cief Method ice280 Analyzer Detector: Camera in UV + - Outlet H + OH - IEF Column Inlet Capillary Focused Zones Dialysis Hollow Fiber Light Beam at 280 nm Sample injection
pi Determination Using cief н Ideal Conditions pi markers with accurate pi values Linear ph gradient created by used carrier ampholytes (usually a single carrier ampholyte is used)
Ideal Conditions н Determining Markers pi Values 15 synthesized peptides are used as the pi markers (Electrophoresis, 21, 603(2000)) pi values of the markers are measured by measuring ph along IEF gel after IEF of the markers Measurement of ph values on IEF gel Anodic ph meter With micro probe Cathodic
Ideal Conditions н Determining ph Linearity for Carrier Ampholytes pi value 10 9 8 7 6 5 4 3 Pharmalyte 3-10 0 500 1000 1500 2000 Peak position (pixel) pi value Servalyt 2-11 11 10 9 8 7 6 5 4 3 2 0 500 1000 1500 2000 Peak position (pixel) 10 Ampholine 3.5-9.5 10 Biolyte 3-10 9 9 8 8 pi value 7 6 pi value 7 6 5 5 4 4 3 0 500 1000 1500 2000 Peak position (pixel) 3 0 500 1000 1500 2000 Peak position (pixel)
Ideal Conditions н Determining ph Linearity for Carrier Ampholytes 10 9 Pharmalyte 3-10 10.5 10 Pharmalyte 8-10.5 pi value 8 7 6 5 r 2 =0.997 r 2 =0.998 r 2 =0.999 pi value 9.5 9 r 2 =0.999 4 8.5 3 0 500 1000 1500 2000 Peak position (pixel) 8 500 1000 1500 2000 Peak position (pixel) 5 4.5 Pharmalyte 2.5-5 8 7.5 Pharmalyte 5-8 pi value 4 3.5 r 2 =0.995 pi value 7 6.5 6 r 2 =0.993 3 5.5 2.5 0 500 1000 Peak position (pixel) 5 300 800 1300 1800 Peak position (pixel)
Accuracy in pi Determination under Ideal Conditions If The ph gradient linear correlation coefficient r 2 > 0.99, 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-r 2 )) 1/2 =(2*(1-0.99)) 1/2 =0.14 Syy=Σ(pI i -pi) 2 pi is the expected pi value of this marker if the ph gradient is linear
pi Determination under Ideal Conditions н Example Sample: Mab1 10 The pi value of this Mab is known to be in ph 5.5 н 7 region The ph gradient of the used carrier ampholyte (Pharmalyte 3-10) has good linearity around ph 5.3 н 7.3 region (r 2 =0.999) pi 9 8 7 6 r 2 =0.999 in pi 5.3 н 7.3 range 5 4 3 0 50 1 0 150 20
pi Determination under Ideal Conditions н Example (Cont d) 7.27 Sample: Mab1 5.31 Mab1 Run the Mab with two pi markers in the linear ph region To achieve optimal accuracy, select the two pi marker peaks that bracket the unknown sample peaks pi value of the Mab is calculated using the pi values of the two pi markers* The pi of the major peak of this Mab is 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 Determination Using cief н Compared to Calculation pi values measured by cief is based on surface charges of a protein In the calculation, the actual folding pattern of the protein is not taken into account We have found that the pi values obtained from cief under denatured conditions (unfolded protein) are closer to that of calculated ones
pi Determination Using cief н Compared to Slab Gel IEF Different pi markers and carrier ampholytes used in cief (usually small molecule markers) and gel IEF (usually proteins markers) may create differences Protein markers usually have multiple peaks In slab gel IEF, matrix effect on ph gradient is usually not compensated unless internal pi markers are applied in samples
pi Determination in Carrier Ampholyte Mixture (Non-Ideal Condition) The ph gradient created by carrier ampholyte mixture is usually non linear: Bigger error may involve in pi determination The accuracy is difficult to estimate
pi Determination in Carrier Ampholyte Mixture - Example pi markers in 2% ph2.5-5 Pharmalyte and 2% ph3-10 Pharmalyte 0 500 10 150 2000 9 8 7 pi value 6 5 4 3 0 500 1000 1500 2000 Peak position (pixel)
pi Determination in Carrier Ampholyte Mixture н 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 pi markers in 2% ph2.5-5 Pharmalyte and 2% ph3-10 Pharmalyte 0 500 10 150 2000 Sample A in 2% ph2.5-5 Pharmalyte and 2% ph3-10 Pharmalyte 0 500 10 150 2000
pi Determination in Carrier Ampholyte Mixture н Example (Cont d) The sample e-gram is stretched and aligned to the marker standard e-gram (acts as a ruler) 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 0 500 10 150 2000 Sample 0 500 10 150
Sample Peak Identification in cief Identify the same peaks in different samples 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 relative retention time in chromatography The identification can be performed regardless of ph gradient linearity (single carrier ampholyte and carrier ampholyte mixture can be used) Only two pi markers are needed to perform the identification
Sample Peak Identification н Need for Internal pi Markers Different matrices Peak may shift in some matrices Different sample concentration Different salt concentration due to different dilution factor Salts squeeze ph gradient created by carrier ampholytes 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 Identification in Single Carrier Ampholytes н Procedure Run all samples with the same two pi markers pi marker peaks should bracket the sample peaks Measued pi values of all sample peaks are calculated using the pi values of the 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 н 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 The measured pi values are used to identify the sample peaks
Sample Peak Identification in Single Carrier AmpholyteнExample In ph 4 н 7 Servalyt 7.5 mm PBS pi marker 5.3 pi marker 6.6 Peak pattern is squeezed by salts 5.96 6.01 15 mm PBS 5.89 6.11 6.21 25 mm PBS 5.91 5.97 6.02 6.12 6.22 Position 5.91 5.97 6.02 6.12 6.22 Sample peaks are identified by their measured pi values Measured pi Peak position differences > 0.03 pi (measured value) can be identified
Sample Peak Identification in CarrierAmpholyte Mixture н Procedure 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 Overlay e-grams of all samples by aligning the two pi marker peaks The sample peaks are identified by their positions in the overlaid e-grams
Sample Peak Identification in Carrier Ampholyte Mixture н Example Sample A in different salt concentrations Sample in 2% ph2.5-5 Pharmalyte and 2% ph3-10 Pharmalyte pi marker 9.50 pi marker 3.78 pi marker 9.50 pi marker 3.78
Sample Peak Identification in Carrier Ampholyte Mixture н Example (Cont ) Overlay and align the two e-grams by the two pi marker peaks Sample in 2% ph2.5-5 Pharmalyte and 2% ph3-10 Pharmalyte pi marker 9.50 pi marker 3.78 pi marker 9.50 pi marker 3.78 Sample peaks are identified by their positions in the e-grams