Phospho-Mania

INITIAL EFFORTS

That's not to say the old standards have died out; many groups still rely on the isotopic labeling approach because it is reliable and easy to use. "P-32 is still the most useful general-purpose reagent out there," says Peter Kennelly, professor of biochemistry at Virginia Tech in Blacksburg, Va. He adds that the reagent is sensitive, versatile, and quantitative. And, it is relatively inexpensive, requiring only basic laboratory equipment.

For laboratories familiar with the method and not averse to radioactivity, P-32 labeling is still frequently the technique of choice for detecting phosphorylation sites. "We still use P-32 quite often because we're good at it, and it's even more sensitive than mass spec right now. But it does require using large amounts of radioactivity that many people shy away from," says Hunter.

As an alternative approach, several companies offer safer modification-specific stains. The GelCode Phosphoprotein Staining Kit from Pierce Biotechnology in Rockford, Ill., is used for direct staining of proteins in SDS-PAGE gels. The Pro-Q Diamond LC Phosphopeptide Detection Kit from Molecular Probes (a division of Invitrogen) in Eugene, Ore., uses a fluorescent dye for liquid chromatography separations. "There are applications, even for those of us who use P-32 – specifically, whole-cell labeling where you have to use prohibitively large amounts of radioactivity – where using these stains for proteomics approaches to identify targets is something we're going to be exploring," says Kennelly.

BE SPECIFIC

Whether radioactive or not, these labeling methods provide a coarse-grain assessment of phosphorylation patterns. Seeing the finer detail, that is, which of the sites are phosphorylated, requires further analysis. The key reagents in such tests are site-specific antibodies, which can be used in such methods as western blotting, immunostaining, ELISA (enzyme-linked immunosorbent assay), and flow cytometry. "Being able to make antibodies to specific sites really revolutionized the field," says Erik Schaefer of Biosource International, Camarillo, Calif.

Western blotting traditionally examines one protein at a time, though it is possible to examine two proteins simultaneously using differently labeled secondary antibodies. For higher levels of multiplexing, though, a growing number of researchers are moving to bead-based assays, in which each bead acts like the well of a microtiter plate.

A number of companies sell kits based on the Luminex xMAP system, including Bio-Rad Laboratories in Hercules, Calif., whose Bio-Plex assays can detect as many as 11 different phosphorylated kinases, a far cry from xMAP's theoretical capacity of 100 different assays at once. But the beads also require a dedicated reader. On the other hand, BD Biosciences in San Jose, Calif., has the Cytometric Bead Array, which can be used with any manufacturer's flow cytometer.

Even higher multiplexing capability is on the horizon. In the fall, Biosource plans to launch a phosphoarray capable of measuring as many as 3,800 signals on four slides. The array will feature capture antibodies (typically monoclonals that recognize both phosphorylated and nonphosphorylated forms of a protein) directly linked to the slide surface. "Developing the array signal allows one to measure both site-specific phosphorylation of the target protein and the total amount of target," says Schaefer.

But antibody-based assays are only as good as the antibodies themselves. "Not all [antibodies] are characterized to the same extent," says Schaefer. Data that look great can be flawed if, for example, an antibody cross-reacts with other sites. "The field has really recognized that we have got to make sure that these reagents are really having the specificity that is required, so that the interpretation is correct," Schaefer adds. "And that there has to be a high enough affinity to really give you the appropriate signal-to noise ratio. Otherwise you end up looking at a lot of background noise."

MASS SPEC ON THE RISE

Some researchers detect phosphorylation events using mass spectrometry rather than antibodies. Mass spec methods generally involve proteolytic digestion of phosphoproteins, followed by tandem mass spectrometry analysis of the resulting fragments to identify phosphorylated sites, which are readily detectable by a change in mass-to-charge ratio compared to unphosphorylated peptides.

Success depends on careful sample preparation, however. "One of the problems is that often phosphorylation stoichiometry is low and so you need ultrasensitive methods or methods for enriching for phosphopeptides from a mixture of peptides, and that's where the technologies are slowly getting better," says Hunter.

Common enrichment methods include IMAC (immobilized metal-ion affinity chromatography) and strong cation-exchange chromatography. Laurence Brill, principal investigator for proteomics at the Genomics Institute of the Novartis Research Foundation, San Diego, and colleagues used IMAC enrichment to identify more than 60 phosphopeptides in a single sample, with an emphasis on tyrosine phosphorylation.¹ Chemical derivatization of phosphate groups also has been used for analysis of phosphorylated residues.

Some researchers use mass spectrometry comparatively, for instance to compare the abundance of a phosphorylated peptide under two different conditions. The keys to such analyses are isotopic-labeling methods such as ICAT (isotope-coded affinity tag), SILAC (stable isotope-labeled amino acids in cell culture), and iTRAQ, from Applied Biosystems of Foster City, Calif.

In ICAT two samples are individually labeled on cysteine residues with so-called isobaric tags, that is, they have identical masses but different isotopic compositions. The samples are then combined, separated, digested, and mass analyzed. The iTRAQ reagents are conceptually similar but can be used to analyze up to four samples at once and, because they are amine-specific, can label any peptide. In SILAC, the cells are cultured in the presence of either a heavy or light form of an essential amino acid prior to protein extraction.

FURTHER PROGRESS

Despite these improvements, mass spec techniques are still problematic. IMAC columns, for instance, select not only phosphorylated peptides, but also acidic peptides in general. Peptide methylation can decrease nonspecific binding, but IMAC is still far from perfect.

For example, IMAC is not always effective at capturing rare phosphorylated species such as phosphotyrosine, which represents less than one percent of the phosphoproteome, says Michael Melnick, vice president for business and corporate development at Beverly, Mass.-based Cell Signaling Technology. "So when you use the IMAC method, you're basically just looking at serine and threonine phosphorylation," Melnick says. IMAC also shows a pronounced bias for multiply phosphorylated peptides, he adds, which further skews the resulting data.

Typical protocols for phosphotyrosine enrichment involve antiphosphotyrosine immunoprecipitation followed by tryptic digestion, IMAC, and tandem mass spectrometry. The resulting spectra contain both phosphorylated and nonphosphorylated peptides, but Cell Signaling Technology has developed a new technique that selects only phosphotyrosine-containing ones. Called PhosphoScan, the method involves immunoaffinity purification of phosphotyrosine peptides directly from a tryptic digest, followed by tandem mass spec. The company is extending the method to functional subsets of phosphoserine/threonine sites, according to Melnick, using antibodies that correspond to consensus phosphorylation sites for different kinase subfamilies.

Another problem with mass spectrometry is that many phosphorylated proteins are difficult to volatilize with high efficiency. "It's not as simple and as straightforward as some people think right now. It still takes some skill and luck," says Kennelly. Proteins that do volatilize frequently suffer loss of phosphate groups, particularly at serine and threonine residues, when collision-activated dissociation is used as a means of producing ions for tandem mass spectrometry. "That makes getting peptide backbone cleavage much more difficult, and therefore sequence information of the peptide more difficult," says Brill.

To overcome this problem, Don Hunt and colleagues at the University of Virginia, Charlottesville, recently developed a technique called electron transfer dissociation mass spectrometry.² In this method, singly charged anthracene anions donate electrons to the peptide of interest and thereby induce fragmentation, with minimal or no loss of phosphate groups and other posttranslational modifications. Though the technique is not yet available commercially, it has generated excitement in the proteomics field. "We are eagerly awaiting its availability," says Brill.

In the end, though, all the above-mentioned techniques represent starting points: After a phosphorylation site is identified, follow-up experiments are required to make sure it is biologically relevant. "The more sites you can identify, the better, but they really require following up. Not that elucidating phosphorylation sites is not valuable," says Brill, "but you really need to find out which ones are more important to the cellular behavior."

Aileen Constans aconstans@the-scientist.com