Sequential multiplex analyte capturing for phosphoprotein profiling

Abstract

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity.

Fig. 1.

Sequential multiplex analyte capturing. Magnetic suspension bead array assays can be performed sequentially, reusing the same sample material (indicated by the blue arrow). The use of a magnetic particle handler enables the quantitative transfer (black arrow) of the magnetic beads from the sample well into the wells containing washing solutions or other assay reagents. Magnetic beads from the first bead array panel are incubated with the samples to capture their respective analyte. Then the magnetic beads are subjected to washing and detection steps and are finally transferred into the readout plate (first row). After retracting the magnetic suspension bead array of the first assay panel from the sample, a bead array from the second assay panel is added and processed as described above but using different detection antibodies (second row). A third bead array assay panel can be applied after removing the second panel (third row) and so on.

Fig. 2.

Ambient analyte theory: repetitive measurement of biotin-PE from same sample. The diagram shows the results for five sequentially performed analyses of the same protein from the same sample from the same well. A model assay using biotin-PE and a biotin-specific capture antibody was used. Anti-biotin antibody-coated magnetic beads were incubated with biotin-PE at five different concentrations (300, 80, 20, 5, and 1 pm) in triplicates. After a 30-min incubation period, the beads were removed from the sample, and new antibody-coupled magnetic beads were added. This procedure was repeated four times, and the assay was read out on a Luminex 100 reader. No analyte limitations were detectable over the five measurements. The data from the first measurement were used for a five-parametric logistic fit. Using the parameters of this fit, the raw assay data were transformed to analyte concentrations (see Table III). After five assays, an analyte decrease of 20% was observed in total, suggesting a captured amount of less than 5% per assay.

Fig. 3.

Repetitive measurements of seven receptor tyrosine kinases from same sample. The diagrams show the results of two sequentially conducted multiplex sandwich immunoassays for the EGFR (A), HER2 (B), HGFR (C), IGF-1R (D), PDGFRβ (E), VEGFR2 (F), and Tie-2 (G) using the same dilution series. Antibody-coupled beads were incubated with recombinant standards at seven different 4-fold dilutions starting for EGFR, HER2, and PDGFβR at 20,000 pg/ml; for IGF-1R and VEGFR at 100,000 pg/ml; for HGFR at 50,000 pg/ml; and for Tie-2 at 10,000 pg/ml. A mixture of biotinylated antibodies specific for the respective RTK, together with SAPE, was used to detect the analytes in the readout system (Luminex 100). The median fluorescence intensities (MFI) of the two sequential assays were plotted against each other and fitted linearly. Standard deviations were calculated from three technical replicates. The table (H) contains slopes, intercepts, and correlation coefficients of the fits for the seven assays. The calculated correlations and slopes close to 1.000 confirm the hypothesis that the analyte can be analyzed twice without changing the concentration of the analyte itself. AU, arbitrary units.

Fig. 4.

Sequential measurement of EGFR and tyrosine phosphorylation of EGFR from same sample. An EGF-stimulated A431 cell line was used for the sequential measurement of EGFR and the generic tyrosine phosphorylation of EGFR. The median fluorescence intensities (n = 3) obtained in two experiments are plotted in the diagram. The left panel shows the results for the experimental order EGFR followed by tyrosine phosphorylated EGFR measurement, and the right panel shows the inverted order. The results do not differ significantly. AU, arbitrary units.

Fig. 5.

Phosphorylation analysis of treated A431 cell line using sequential multiplex analyte capturing. A431 cell cultures were treated with EGF, PMA, and a combination of sodium vanadate and hydrogen peroxide to give different responses in receptor signaling. First, nine different color-coded beads (assay panel 1) conjugated with antibodies specific for total EGFR and EGFR phosphorylation at threonines 654 and 669; serine 1047; and tyrosines 845, 1045, 1068, 1086, and 1173 were incubated with the protein extract sample overnight. To carry out the detection, the beads were then removed from the sample using a magnetic bead handling robot and added to a solution of biotinylated EGFR-specific antibody followed by a third incubation step with SAPE. After removing the beads of panel 1 from the sample, antibody-coupled beads from assay panel 2 (seven RTKs: EGFR, HER2, HGFR, IGF-1R, PDGFRβ, VEGFR2, and Tie-2) were added and incubated with the sample overnight. Generic phosphorylation was detected by retracting the beads from the sample followed by incubation with a biotinylated tyrosine phosphorylation-specific antibody and SAPE. In the final analysis, the antibody-coupled beads from assay panel 3 were added to the same samples and incubated overnight. The relative abundance of the RTKs was detected by incubation with an antibody mixture of seven different biotinylated antibodies specific for the respective RTK. The figure shows the median fluorescence intensities (n = 3) for the sequentially performed assay panels.