Microchip Electrophoresis Assay for Calmodulin Binding Proteins

Abstract

The calcium signaling protein calmodulin regulates numerous intracellular processes. We introduce a sensitive microchip assay to separate and detect calmodulin binding proteins. The assay utilizes an optimized microchip electrophoresis protein separation platform with laser-induced fluorescence detection. Fluorescence-labeled calmodulin modified with a photoreactive diazirine crosslinker allowed selective detection of calmodulin binding proteins. We demonstrate successful in-vitro crosslinking of calmodulin with two calmodulin binding proteins, calcineurin and nitric oxide synthase. We compare the efficacy of commonly applied electrophoretic separation modes: microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography/gel electrophoresis, and nanoparticle colloidal arrays. Out of the methods tested, polydymethylsiloxane/glass chips with microchip zone electrophoresis gave the poorest separation, whereas sieving methods in which electro-osmotic flow was suppressed gave the best separation of photoproducts of calmodulin conjugated with calmodulin binding proteins.

Keywords: calmodulin binding protein; colloidal array; micellar electrokinetic chromatography; microchip electrophoresis; photo-crosslinking.

Figure 1.. Detection of photochemically crosslinked CaM-CN and CaM-eNOS by NHS-LC-SDA.

SDS-PAGE (left) and western blot (right) showing photoproducts of CaM-CN (~77 kDa) in lane 5 and CaM-eNOS (~153 kDa) lane 6.

Figure 2.. CZE of CaM-CBP conjugates.

Electropherograms of (A) CaM-eNOS and CaM-AF647; (B) CaM-CN and CaM-AF647; (C) a mixture of CaM-eNOS and CaM-CN. Peaks 1, 2, and 3 are CaM-eNOS, CaM-CN, and CaM-AF647, respectively. The field strength was 241.9 × 10³ V/m with a 0.31-m bare silica capillary (id 50 μm), BGE: 75 mM boric acid, pH 9.2, 3.5 mM SDS, 0.05% (m/v) HPMC.

Figure 3.. Microchip electrophoresis of CaM-CN and CaM-eNOS photoproducts with a 0.10-m serpentine glass chip.

(A) Mixture of CaM-AF647 (~150 nM), CaM-CN photoproducts (~100 nM), and AF647 (10 nM). Peaks 1 and 3 are assigned to CaM-CN photoproducts, peak 2 to CaM-AF647, and peak 4 to AF647. (B) Mixture of CaM-AF647, CaM-eNOS photoproducts, and AF647. Peaks 1 and 3 are assigned to CaM-eNOS photoproducts, peak 2 to CaM-AF647, and peak 4 to AF647. For both separations the buffer composition was 50 mM boric acid, pH 9.2, 3.5 mM SDS, separation potential 7.5 kV, injection time 0.3 s. The detection length was ~0.08 m.

Figure 4.. Separation of CaM-CN and CaM-eNOS photoproducts with a PDMS/glass chip and EOF suppression.

(A) Electropherograms for a series of repeated injections. A simple “T” PDMS/glass chip with a 0.035-m separation channel was used. Peak 1 was identified as CaM, peaks 2 and 3 as CaM-CN photoproducts, and peak 4 as CaM-eNOS photoproducts. Consecutive multiple runs (n=5) show consistent migration times. (B) Apparent mobility vs. relative molecular mass of analytes. The linear least squares fit yields log μ = −0.0010 M_r − 7.98, where M_r is the relative molecular mass. Peak 3 was used for the mobility calculation of CaM-CN. Error bars show the standard error. The separation conditions were as follows: 4X TBE (360 mM tris-borate, 8.0 mM EDTA), pH 8.3, 0.05% (m/v) HPMC, 10 mM SDS, separation potential 800 V, detection length ~0.015 m.

Figure 5.. Separation of CaM-CBP photoproducts in a silica-nanoparticle colloidal array in a PDMS/glass microfluidic device.

The particle diameter was 170 nm, detection length, ~5 mm. (A) Run buffer: 3X TBE, pH 8.5 with 3.5 mM SDS, separation field strength 5.8 × 10³ V/m. Peaks 1, 2 and 3 can be identified with CaM-AF647, CaM-CN photoproduct, and CaM-eNOS photoproduct, respectively. (B) Run buffer: 4X TBE, pH 8.5 with 3.5mM SDS, field strength 4.3 × 10³ V/m, total concentration of the mixture ~60 nM. Peak 1 corresponds to CaM-AF647, peak 2 to both CaM-CN and CaM-eNOS photoproducts, and peak 3 to a CaM-eNOS photoproduct. (C) Apparent log mobility vs. relative molecular mass of analytes for the separation shown in A with 3X TBE, n=3.