Protein Isoforms and their Resolving Applications

Transcription

1 ANALYSIS OF PROTEIN ISOFORMS USING CAPILLARY ZONE ELECTROPHORESIS WITH A DYNAMIC COATING William W. P. Chang, David C. Bomberger, Luke V. Schneider Target Discovery, Inc., 4015 Fabian Way, Palo Alto, CA 94303, INTRODUCTION Changes in protein isoform expression have been correlated with multiple diseases. Resolving these isoforms is often difficult because single amino acid, phosphorylation, and glycosylation patterns affect only a small part of the protein structure. HPLC, isoelectric focusing gel electrophoresis (IEF), and immunochemical approaches have been applied to isoform analysis, but these tend to be laborious or inaccurate due to interferences. Capillary electrophoresis (CE) potentially has many advantages, including speed, low sample/buffer requirement, and ease of automation. We show that capillary zone electrophoresis (CZE) can be a powerful tool for rapid and robust protein isoform separation when electroosmotic flow (EOF) is controlled. We demonstrate reproducible and high resolution protein isoforms separations using fused silica capillaries treated with a new dynamic coating, EOTrol TM LN. MATERIALS AND METHODS All reagents were purchased from Sigma-Aldrich (St. Louis, MO), except human α 1 -antitrypsin (EMD Biosciences, La Jolla, CA). Uncoated silica capillaries were purchased from Supelco (Bellefonte, PA) and were pre-conditioned by rinsing with methanol, HCl, and NaOH, with water rinses in between. DB-WAX capillaries (coated with polyethylene oxide, PEO) were purchased from Agilent Technologies (Wilmington, DE) and were pre-conditioned with methanol and water rinses only. The P/ACE MDQ Capillary Electrophoresis System (Beckman Coulter Instruments, Fullerton, CA) was used in all experiments. All CZE experiments were liquid cooled (at 25 C). UV detection (214 nm) was used in all experiments. Specific experimental conditions are described on the figures. Protein stocks were prepared in HPLC grade water, without denaturation. Aliquots were stored frozen until use. These stock solutions were diluted with the respective CZE buffers prior to use. Blanks were adjusted to the same buffer ion type and strength as the protein samples. 1

2 Analysis of Protein Isoforms Using Capillary Zone Electrophoresis with a Dynamic Coating THE EOTROL SUITE OF DYNAMIC COATINGS EOTrol is a novel suite of acrylamide, N-substituted acrylamide copolymers that serves as dynamic coatings for both CE and microfluidic uses. EOTrol provides the following benefits: Reduce, eliminate or reverse EOF with uniform coating Insensitive to buffer type, ph, chaotraopic agents or surfactants No absorbance above 250 nm 2

3 Analysis of Protein Isoforms Using Capillary Zone Electrophoresis with a Dynamic Coating MISMATCHED EOF = RESOLUTION LOSS Research has shown1 that mismatched EOF, resulting from nonuniform coating, leads to more resolution loss than no EOF reduction (see left panels). This points to the need of having a coating that provides uniform, effective EOF suppression. 1 Herr et al. Anal Chem 2000; 72:

4 BSA ISOFORM SEPARATIONS Figure 1. Bovine serum albumin (69 kd) has three isoforms (pi = 5.4, 5.5, and 5.6). 2 While the PEO coating reduces EOF, this reduction is insufficient to completely resolve the isoforms. Figure 2. Three major peaks are detected in a bare silica capillary using EOTrol LN, which provides more uniform EOF control than a coated capillary and, consequently, higher resolution. Reproducibility is show in the next panel. Figure 3. Bovine serum albumin is a protein widely used in many routine research laboratory procedures. It has three pi isoforms (pi 5.4, 5.5 and 5.6) determined empirically by 2D-PAGE separation. (Adapted from BioRad). 2 Bio-Rad Life Science Research Products Catalog 2000/01, Hercules, CA: Bio-Rad Laboratories, 2000:211. 4

5 Analysis of Protein Isoforms Using Capillary Zone Electrophoresis with a Dynamic Coating TRANSFERRIN ISOFORM SEPARATIONS Figure 1. There are four known human transferrin (76 kd) isoforms in normal individuals. Only three peaks were resolved in the PEO-coated capillary under optimal conditions. Tf isoforms have been correlated with: Chronic alcohol abuse Iron deficient anemia Figure 2. Four peaks are observed using EOTrol LN, showing slightly better resolution than CZE with CEOFix CDT kit (panel to right). Tf isoforms are modified with up to 5 sialic groups with the tetrasialo being the predominant form. Figure 3. Transferrin (Tf) is a plasma protein that carries iron between old blood cells and the bone marrow. The separation shown uses the CEOFIX CDT method (Analyis, Belgium). 3 Normal human serum contains four isoforms with different carbohydrate contents. Numerals indicate number of sialic groups for each isoform. 3 Wuyts B et al. Clin Chem, 47: (2001). 5

6 Analysis of Protein Isoforms Using Capillary Zone Electrophoresis with a Dynamic Coating α1-antitrypsin ISOFORM SEPARATIONS Figure 1. Eight protein bands related to α 1 -antitrypsin (52 kd) have been reported, using isoelectric focusing. 4 Separation in a commercial PEO-coated capillary gave only two partially resolved peaks. Figure 2. With EOTrol LN, up to 8 peaks are seen with 5 peaks well resolved in an bare silica capillary. This separation is far superior than that obtained from a PEO-coated capillary and might suggest isoforms previously unresolved by IEF. Figure 3. α 1-Antitrypsin (α 1-AT) is a major inhibitor of serine proteases found in human plasma. Six isoforms (M1, M2, M4, M6, M7, M8) separated by 2-D PAGE are shown. 5 α 1-AT has been correlated with: Alzheimer sdisease Emphysema Liver disorders 4 Fagerholl MK, Laurell CB. Clin Chim Acta 1967;16: Mills K et al. Clin Chem 2001; 47:

7 POOR RESOLUTION IN UNCOATED SILICA CAPILLARIES Uncoated capillaries, often lead to poor resolution of protein isoforms--or, no resolution at all. This is caused by the strong EOF under most separation ph conditions. This figure shows partial resolution of BSA in an uncoated capillary (Tf and α 1 -AT results are worse). EOTROL ALLOWS REPRODUCIBLE ISOFORM SEPARATIONS! Five consecutive BSA separations were conducted in an uncoated capillary with EOTrol LN added to the run buffer. The run-to-run mobility and peak area of the three peaks resolved are very reproducible. Similar results are observed with Tf and α 1 -AT isoform separations with EOTrol LN. 7

8 Analysis of Protein Isoforms Using Capillary Zone Electrophoresis with a Dynamic Coating CONCLUSIONS EOTrol dynamic coatings provide superier resolution for protein isoform separation. EOTrol makes rapid, high-resolution, and reproducible CZE analysis of protein isoforms an invaluable tool for clinical applications. 8