Antibody Array Analysis with Label-based Detection and Resolution of Protein Size

Eva Boretti,Fridtjof Lund-Johansen,Vaclav Horejsi,Geir Tjønnfjord,Hans-Christian Aasheim,Heidi Slåstad,Daniel de la Rosa Carrillo,Weiwei Wu,Tom Frey

doi:10.1074/mcp.m800171-mcp200

Abstract

Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670-10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclin-dependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes.

Highlights

Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines
All the proteins in the sample are reacted with haptens or fluorescent dyes, and the total amount of protein associated with each capture reagent is measured [9, 10]
In Western blotting, antibody reactivity is resolved against protein size, to detect specific binding and cross-reactivity as discrete bands

Summary

Introduction

Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. High throughput analysis of signaling proteins is possible with the reverse protein arrays described by Petricoin and colleagues [8] In these assays, the samples rather than the affinity reagents are multiplexed, and each array is labeled with a single or two antibodies. In Western blotting, antibody reactivity is resolved against protein size, to detect specific binding and cross-reactivity as discrete bands. Measurement with antibody arrays and labelbased detection is comparable with Western blotting where the lanes have been compressed into a single band. The fact that non-specific bands in Western blots are frequent and sample-dependent is a strong argument against the current analysis platform [6].

Methods

Results

Conclusion