Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings.
Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.
Chang
William W P
WW
Target Discovery, Inc., Palo Alto, CA 94303, USA. william_chang@targetdiscovery.com
Hobson
Christine
C
Bomberger
David C
DC
Schneider
Luke V
LV
D000818:Animals; D020099:Coated Materials, Biocompatible; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D020033:Protein Isoforms / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D014168:Transferrin / Q000302:isolation & purification; D000515:alpha 1-Antitrypsin / Q000302:isolation & purification
D020099:Coated Materials, Biocompatible; D020033:Protein Isoforms; D011506:Proteins; D014168:Transferrin; D000515:alpha 1-Antitrypsin; D012710:Serum Albumin, Bovine