Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Much communication in the biological world takes place by means of phosphorylated protein cascades. Phosphate groups on tyrosines, serines and threonines carry information from membrane-bound receptors through a series of cytoplasmic messengers into the nucleus, for example, ultimately dictating which and whether genes will be transcribed.
Monitoring entire pathways and networks rather than just individual kinase and phosphatase substrates—themselves often kinases and phosphatases—is a powerful way to assess pathology, identify biomarkers and evaluate the action of drug treatments.
Phosphoproteins are most commonly detected by antibodies, either in Western blots or ELISA-like formats. Yet in their traditional forms, these assays do not lend themselves to combining more than a few assays, leading the researcher to seek alternatives. Here we discuss different approaches to multiplexing protein detection.
Not a cytokine assay
Assaying for proteins with specific post-translational modifications (PTMs) requires some special considerations. “With phosphoproteins you need to capture the protein, and then you need to detect the phosphorylated epitope of interest,” says Chris Linnevers, global product manager at Bio-Rad Laboratories. A typical assay will recognize a specific phosphorylated residue, and there may be several of these on a given protein.
Multiplexing such assays leads to concerns about cross-reactivity beyond those typically seen in other protein immunoassays. Vendors publish matrices—and sometimes interactive apps (e.g., Bio-Rad’s Bio-Plex® Assay Builder—to help determine which antibodies can be used in the same assay.
To determine how much of a given site is modified, the signal is compared to that of total protein. But “they can’t be run in the same well, because there’s cross-reactivity that can’t be avoided” – both phosphorylated and non-phosphorylated versions will be picked up by antibodies targeting the total protein, explains Linnevers.
The protocol for assaying secreted proteins like cytokines typically calls for serum or cell-culture medium. But cells need to be lysed to find intracellular phosphoproteins or membrane-bound proteins. And their phosphorylation status must be stabilized by including factors such as kinase and phosphatase inhibitors in the buffer.
Multiplexing
Running many assays in the same space can save labor, time and reagents; increase throughput; and simultaneously answer many questions using a single, limited, precious sample. But colorimetric assays like ELISAs can’t easily yield simultaneous multiple different readouts. The case is similar for radiolabeled or luminescent assays like Western blots.
By far the most common techniques to multiplex immunoassays index the location of each assay either spatially or by distinct fluorescent signal. As with a sandwich ELISA, a primary (or capture) antibody is attached to a solid support; the phosphoprotein is captured by that antibody, and a secondary antibody then recognizes the bound phosphoprotein. The secondary antibodies can be directly labeled with fluorophores or compounds such as biotin, or they can be recognized by a labeled tertiary antibody (to amplify the signal).
In planar arrays, the primary antibodies are directly or indirectly attached to glass, plastic or a membrane in an ordered array. The sample and reagents are applied to the entire array as if it were a single assay, yet each spot functions essentially as a distinct assay with signal intensity increasing with the number of detected epitopes.
In suspension arrays, magnetic or polystyrene beads act as the solid support to which the primary antibody is attached. The beads themselves are dyed with varying amounts of one to three different fluorescent labels, allowing for up to 500 unique combinations—meaning that up to 500 distinct epitopes theoretically can be probed in a single reaction.
Off-the-shelf
Some heavily multiplexed phosphoprotein immunoassays are available off the shelf, but none yet even approaches 100 (let alone 500) analytes.
RayBiotech’s RayBio® Human RTK Phosphorylation Antibody Array, for example, is a glass slide spotted with antibodies against 71 different tyrosine kinase receptors. The assay uses a biotinylated secondary antibody to generically recognize phosphotyrosine, and a fluorescence readout using a microarray scanner. Each glass array is formatted as either four or eight subarrays to run multiple samples. The assay is also available in single-array membrane format with a chemiluminescent readout. “Anything basically that will read a chemi membrane will also work with the membrane-based arrays, including the LI-COR Odyssey system,” notes Valerie Jones, the company’s manager, marketing and technical support.
Many multiplex immunoassays are built on the bead-based Luminex xMAP® system. Bio-Rad’s Bio-Plex Pro™ Cell Signaling MAPK Panel, for example, consists of antibodies against nine different phosphoproteins coupled to different magnetic beads, with a biotinylated secondary antibody recognized by a fluorescent streptavidin-phycoerythrin reporter. The assays are read using either a dedicated flow cytometry-based instrument (like the Luminex® 100/200™ or the Bio-Rad-branded Bio-Plex 200) or a dedicated fluorescent imager-based magnetic bead reader (e.g., the Luminex MAGPIX®).
There is robust competition among Luminex partners to sell assays in major areas where protein biomarkers are key. “This leads to continuous improvements in assay sensitivity as well as product quality, which ultimately benefits researchers,” Linnevers says.
But wait! There’s more!
Competition doesn’t end with off-the-shelf kits, nor with Luminex, either.
Many vendors of multiplex immunoassays let customers purchase individual validated assays to be mixed either by the customer or the vendor. Luminex’s xMAP Kit Finder application lets researchers search by protein for assays from at least seven vendors.
BD Biosciences markets its own fluorescent-bead assays under the BD™ CBA ( “cytometric bead array”) name, which are run on traditional flow cytometers. The company’s Cell Signaling Flex Set System lists reagents specific for a total of 22 phosphorylated proteins that can be mixed and matched into multiplex assays.
Similarly, planar arrays can often be custom designed using either the vendor’s or the customer’s antibodies, and they can sometimes even be manufactured by the customer (depending on the technology). Researchers with access to microarray printing equipment can print their own arrays, for example. But, warns Robert Matson, founder and president of QuantiScientifics, directly immobilizing proteins on a surface is a stochastic process that can lead to variations in spot size and binding efficiency. His company uses an oligonucleotide linker technology to attach the antibodies to pre-printed 96-well plates, and customers can also purchase plates and linker kits to create multiplex assays in their own labs.
Meso Scale Discovery (MSD) spots patterned antibody arrays onto custom 96-well plates but uses a proprietary electrochemiluminescent technology as a readout. Many phosphoprotein assays are available for the platform.
Immunoprecipitation for mass spec
Another multiplexing option—and one that is relatively unbiased in terms of what it finds—is mass spectrometry.
One approach relies on purification via immobilized metal affinity chromatography. In this strategy, researchers process their samples using metal affinity beads to enrich for phosphopeptides, then analyze those by liquid chromatography-coupled mass spec.
Cell Signaling Technology's enrichment method of choice is immunoaffinity purification, which can selectively enrich for low-abundance phosphopeptides. “Metal affinity enrichment is non-discriminant and driven by the presence of more abundant proteins, so your subsequent LC-MS/MS analysis can miss the less-abundant phosphopeptides,” explains Matt Stokes, a scientist in CST's proteomics services group.
The company offers two services: PTMScan® Discovery kits and services use antibodies specific to a particular PTM (such as phosphotyrosine, phosphoserine and phosphothreonine) to enrich peptides prior to LC-MS/MS analysis. PTMScan Direct services focus on targets within a limited regulatory network rather than a general class of PTMs, using site-specific antibodies to “protein PTMs within a common signaling pathway or [that are] among the same class, like kinases,” he says. ”Basically, this is like a more efficient and quantitative way of doing hundreds of Western blots.”
Clearly, there’s no shortage of multiplexing options for phosphoprotein analysis. Some are open and flexible platforms running on readily available equipment, others are more proprietary and require “heavy entry fees to get in on the platform,” notes Matson. There are also the usual considerations of cost, speed, throughput, sensitivity, specificity, comfort level and familiarity. But however it’s done, the benefits of extracting so many answers from a single, small sample surely make it worth a try.
Image: Luminex Corp.