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 antibodycoated, 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. Molecular & Cellular Proteomics 9:2474 -2481, 2010.Phosphorylation of proteins is an integral part of the signal transduction of eukaryotic cells as it modulates the activity of complex protein networks. Although Western blot-and immunoprecipitation-based MS approaches (1, 2) can lead to detailed insights into these processes, most of the integrated approaches only allow a static view of protein phosphorylation because they are not suitable for the screening of hundreds of samples. Either planar or bead array-based sandwich immunoassays can be used to analyze the quantity and activation state of signaling molecules in multiplex, enabling the systematic profiling of protein abundance and post-translational modifications (3-6) in hundreds of samples. However, multiplex immunoassays are only suitable for the simultaneous analysis of a limited number of proteins. The detection of comprehensive phosphorylation patterns is difficult as this involves assay systems that are incompatible with multiplexing.In principle, two sandwich immunoassay setups are possible for probing the phosphorylation state of a protein. The first setup applies a capture antibody specific for a non-modified part of the protein and uses a phosphorylation state-specific detection antibody. When applied to an array-based format, however, this setup does not allow for the simultaneous measurement of the abundance and the degree of phosphorylation (3, 4). A mixture of detection antibodies, one specific for the phosphorylation site and one specific for the nonmodified site of the protein, would bind simultaneously to the two different epitopes, and assay signals could not be further deconvoluted by the spatial or color code of the array. The second sandwich immunoassay setup for the analysis of protein phosphorylation applies a phosphorylation state-specific capture antibody and a protein-specific detection antibody. In such a setup, an anti-phosphotyrosine antibody (e.g. mAb 4G10) cannot be applied as a capture antibody because a huge variety of tyrosine phosphorylated proteins would be captured, and spe...

A Dual Array-Based Approach to Assess the Abundance and Posttranslational Modification State of Signaling Proteins

“…Although we describe how to perform the process using a magnetic plate separator and manual washing in a 96-well plate, it is also possible to use a robot—for example, a magnetic bead transfer system (King Fisher, Thermo Fisher Scientific), which facilitates semiautomated washing and incubation (24).

Combine the coated beads in Bead Array Assay Buffer I (Recipe 16) so that there are 2000 beads per bead population in 20 µl of fully mixed beads.

Mix the beads by vortexing the diluted beads.

Adjust the protein concentration in each cellular sample so that each sample has the same protein concentration and keep the samples on ice.

Note: We prepare our samples for analysis of β -catenin at 10 to 25 µ g of total protein per well.

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Snapshots of Protein Dynamics and Post-translational Modifications In One Experiment—β-Catenin and Its Functions

“…When very small amounts of capture antibody are immobilized on a microspot or a microbead, so little of the target analyte is captured that the analyte concentration in the remaining solution is virtually unchanged. As such, multiple post-translational modifications from the same protein in the same sample can be measured in parallel without the fear of competition (22).…”

Targeted protein-omic methods are bridging the gap between proteomic and hypothesis-driven protein analysis approaches

“…In this new approach, magnetic bead-linked antibodies are incubated in sequential fashion with cell lysates in the solution-phase. Then, following magnetic capture, these beads are incubated with secondary antibodies and quantified via a bead-based flow sorting machine [55]. …”