Isoelectric focusing of proteins in silica packed capillaries and channels

Brooke M Koshel, Purdue University

Abstract

One of the goals of proteomics is to discover protein biomarkers, which are quantitative indicators of biological processes, disease progression, and drug therapy response. Protein biomarkers are expected to exist in low ng/mL concentrations in the early stages of disease, and their detection is hindered by high abundance proteins and a high dynamic range of concentrations, spanning several orders of magnitude. A widely used method for protein characterization is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), where isoelectric focusing (IEF) separates proteins based on isoelectric pH in the first dimension, followed by sieving electrophoresis in the second dimension, which separates proteins based on molecular weight. IEF is carried out in commercially available gel strips having an immobilized pH gradient, and when combined with electrophoresis, the resulting separation is completely orthogonal. Some of the drawbacks of these gels are their detection limit, inability to cover the proteome dynamic range, and the lengthy amount of time required in sample preparation as well as the focusing process itself. 2D-PAGE as a whole also suffers from the ability to identify proteins, because the peak capacity is a few hundred whereas there are tens of thousands of proteins in blood serum. Removal of proteins from the gel by digestion for analysis via mass spectrometry (MS) is a labor intensive process. For these reasons, protein analysis has shifted from gels to capillaries. IEF is reduced from hours to minutes in both sample preparation and analysis time, and capillaries are more readily integrated with MS. Capillary isoelectric focusing (cIEF) is conducted in open tubes, generally having a wall modification to reduce protein adsorption and electroosmotic flow. Remobilization of the proteins for MS analysis is accomplished either chemically by changing the pH or with pressure. Both require the use of an electric field during remobilization. Herein, capillaries packed with 900 nm silica particles modified with a brush layer of polyacrylamide are explored for cIEF, and their performance is compared to open capillaries having the same modification. A standard protein mix containing trypsin inhibitor, carbonic anhydrase, and myoglobin is focused and remobilized using pressure in the absence of an electric field. Resolution was determined to be 0.1 pH units after remobilization. Minimal adsorption was observed. The packed capillaries described above point to an opportunity for combining biomarker discovery with clinical validation, which requires hundreds or thousands of trials. Clinical validation is the bottleneck in the biomarker discovery pipeline. The concept of silica packed capillaries was extended to silica packed channels, which can more readily be developed into an array format. IEF of a model protein is demonstrated in a three channel format, showing protein shifts from channel-to-channel of only 200 to 300 μm. IEF in a 1 cm long channel yielded similar results to IEF in a 1.65 mm long channel, indicating that multiple, short channels in an array format is feasible. Finally, IEF of clinically relevant samples, prostate specific antigen (PSA) and a monoclonal antibody, was carried out and compared to IEF using a gel strip.

Degree

Ph.D.

Advisors

Wirth, Purdue University.

Subject Area

Analytical chemistry|Biochemistry|Physical chemistry

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