by Jeffrey M. Perkel
Though often modeled in isolation, cells are social creatures. Like a teenager with unlimited texting, they send and receive a steady stream of molecular status updates: OMG, did u smell that carbon source? LOL!!!
Those messages are routed intracellularly along signal transduction circuits that yield changes in gene expression patterns and, ultimately, in behavior, morphology, or phenotype. At the heart of most such circuits is a series of protein switches that are toggled by the addition or subtraction of phosphate groups. The enzymes that catalyze these reactions are called kinases and phosphatases, and they play critical roles in both development and disease. Fortunately, a wide array of molecular tools exists to study these processes, many of them based on phospho-specific antibodies.
As the name suggests, phospho-specific antibodies are simply antibodies that target a specific phosphorylated form of a protein. Useful in assays ranging from Western blots to immunohistochemistry, and from ELISA to flow cytometry, these antibodies have become the cornerstone of cell signaling research, and literally thousands now exist. Cell Signaling Technology offers more than 800 phospho-specific antibodies, representing hundreds of different phosphorylation sites, according to Ze'ev Gechtman, the company's group leader for molecular assays. PhosphoSolutions offers nearly 175, most of which target proteins of interest to neuroscientists, according to president Michael Browning.
Phospho-specific antibodies can be as generic as a pan-phosphotyrosine antibody, or as discriminating as an antibody specific to a given phosphorylation site. Among PhosphoSolutions' portfolio is a pair of antibodies that can distinguish two different phosphorylated forms of aquaporin2; one is specific to the protein form phosphorylated on Ser261, the other, on Ser264. Between these two extremes are what Cell Signaling Technology calls motif-specific antibodies—that is, they recognize classes of molecules based on the kinase (or family of kinases) that modified them. One such antibody, for instance, recognizes serines that were phosphorylated by CDKs, in the context of the motif (K/R)(S*)PX(K/R).
Whatever the specificity of the antibody, to a large extent customers apply them to the standard suite of immuno-assays—Western blots, ELISA, immunohistochemistry, and so on. According to Browning, the assay to which most customers apply his company's antibodies is the Western blot. "I would say it's like 80/20, Western blotting versus immunohistochemistry or other assays," he says. At Symansis, a two-year-old biotech firm based in New Zealand, the company's focus is signaling ELISAs, an application the company supports with a twist.
ELISA plates are usually manufactured using a single capture antibody—that is, each well contains the same capture antibodies. Thus, if a user wanted to test a lysate for two different analytes, he would have to test those on two different plates. Symansis, though, offers a more flexible alternative called the ELISA array, in which users mix and match ELISA well "strips" that they can lock into a microtiter plate-sized "form" to build a custom assay.
According to Lyndon Foster, director of sales and tech support for Symansis USA, this approach provides flexibility in assay design, consistency (the color-coded strips are stable for up to 2.5 years in the refrigerator), and speed. Customers can assay as many as eight different analytes at once per plate in this format, in the same amount of time and with the same effort as they normally would expend on just one analyte. "The customer can get data on multiple kinase targets in one run of the kit, as compared to having to run individual kits for each one of the targets [in the kit]," he says.
EMD Millipore and Thermo Fisher Scientific support an intracellular version of the ELISA, called the "In-Cell ELISA." ELISA, explains Matt Baker, business development director for proteomics life science research at Thermo Fisher Scientific, is an assay best suited for soluble protein. But many signaling molecules are either associated with, or inserted in, the cell membrane itself. "It can be more difficult to probe those types of samples because for an ELISA to work you basically have to have things in solution," he says.
In-cell ELISA eliminates this problem basically by circumventing the sample preparation step. Cells are simply grown on the bottom of the ELISA plate, fixed and permeabilized in situ, and then incubated with detection antibodies and read. Both Thermo and EMD Millipore support two-color versions of this assay, in which one fluorescent color is used to quantify total protein (say, all Akt kinases) and another for a specific phosphorylated variant (say, Akt-pSer473). EMD Millipore's assays are called "Dual Detect CELISA Assay Kits"; Thermo's are "In-Cell ELISA Kits", and they are, according to Baker, compatible with "a lot of different systems," including LI-COR 's Odyssey, which offers two-color detection of near-infrared fluorophores for what it calls "In-Cell Westerns," "On-Cell Westerns" (basically, an In-Cell Western without the permeabilization step), and "In-Gel Westerns."
Of course, analyzing one or even two proteins at a time is too low a throughput to effectively study an entire signaling pathway; cell-signaling cascades can involve dozens of proteins. "That's the main stumbling block in the field, is the inability to do multiplex [assays] with antibodies," says Browning.
There are a few multiplexed signaling assays available, however, and more are coming. One approach is the multiplexed bead-based technology developed by Luminex, available from a wide array of vendors.
Alternatively, Cell Signaling Technology last year launched an antibody array that probes the phosphorylation state of 39 separate proteins in cell lysates on up to eight subarrays on a single microscope slide. Available in both fluorescent and chemiluminescent forms, the PathScan RTK Signaling Antibody Array queries 28 receptor tyrosine kinases and 11 intracellular signaling "nodes" using sandwich immunoassays, says Gechtman.
The approach, says Gechtman, can save researchers considerable time and effort. "If you compare to how many Western blots would one need to do to get the same amount of data," he says, "[it] is really close to the human capacity of what people can do using the old-fashioned technique."
(PhosphoSolutions is also developing an antibody array targeting key synaptic signaling molecules, and hopes to have it available by next year's Society for Neuroscience conference, according to Browning.)
Most of the above-mentioned assays, with the exception of in-cell ELISAs, probe signaling markers in cell or tissue extracts, as opposed to in situ. Some assays, though, can provide a cell-by-cell assessment.
EMD Millipore's FlowCellect™ kits use flow cytometry to study the activation and cross-talk of major cell signaling pathways and measure downstream biological effects. These kits typically include three or four "key [signaling] decision points" to probe molecular circuits, says Jason Whalley, flow cytometry marketing manager at EMD Millipore. For instance, the FlowCellect PI3K/MAPK Dual Pathway Activation & Cancer Marker Detection Kit uses antibodies against phosphorylated ERK (an indicator of MAPK signaling), phosphorylated Akt (an indicator of PI-3K signaling), and Ki67 (a measure of cell proliferation).
This three-marker kit "can give a very easy and clear answer of which pathway is activated by the upstream component, and also what is the consequence of the pathway activation as well as cross-talk," explains Matthew Hsu, the company's director of R&D for advanced cellular technology.
Other cell-based assays include label-free platforms such as Corning's EPIC system, and Thermo Fisher Scientific's Cellomics product line. Cellomics high-content screening assays, read on the company's ArrayScan instrument, detect not only protein levels, but also their spatial distribution, in a 96-well format, according to Baker. The Cellomics Phospho-p53 and p53 Activation Kit, for instance, measures both phosphorylated and unphosphorylated p53 using phosphorylated antibodies, as well as its nuclear and cytoplasmic distribution (and overall cell count and morphology) using a DNA stain (Hoechst).
Naturally, these assays are only as good as the antibodies that make them work. Thousands of phospho-specific antibodies are commercially available, yet antibodies remain a persistent problem in the cell-signaling field. "I think most people would agree these platforms should work if we could just get antibodies that really were specific," says Browning.
Biotech firms continue fleshing out their product portfolios. But it's a Herculean task; according to Baker, perhaps a quarter or more of all human proteins are or can be phosphorylated, and many of those sites "are going to mean something."
"It's very difficult to have canned immunoassays to cover all of those phosphorylation sites," he says. But there is another option: "Mass spectrometry allows incredible levels of multiplexing."
Phosphoproteomics has its own problems, of course. And researchers have made tremendous progress using phospho-specific antibodies. Yet, concludes Baker, the future probably belongs to mass spec. "Mass spec is going to be driving a lot of these measurements in the future," he says.