Phosphoproteomics: Miles To Go Before It’s Routine

Abstract

Researchers see major technological advances but still have significant challenges to overcome.Not so long ago, the process for determining protein phosphorylation was a labor-intensive and serial process. Typically, a researcher would use 32 P-labeling coupled with 2D gel electrophoresis phosphopeptide mapping, autoradiography, band excision, and Edman sequencing. In addition to expending much time and effort, researchers had to work with radioactivity, and the method could not be multiplexed. Now in 2009, two complementary techniques for identification of phosphorylation sites, MS and microarrays, have provided significant increases in throughput. In discovery mode, researchers can identify thousands of phosphorylation sites with LC/MS n , and in profiling mode, researchers can detect thousands of phosphorylation events and predict the substrates of various protein kinases with planar, suspension, and reverse microarrays. 1 As the technology has improved and researchers have been able to glean impressive amounts of information from phosphoproteomics experiments, more labs have shown interest. "I think the field of phosphoproteomics is poised to take a big leap forward," says Timothy Veenstra of the U.S. National Cancer Institute. And the widespread enthusiasm among researchers is tangible. "It's probably the most exciting time right now in phosphoproteomics," says Daniel Figeys of the University of Ottawa. " [Phosphoproteomics] used to be kind of stuck a few years ago, where the numbers of phosphorylation sites that could be mapped were dreadfully small," he says.But mapping so many sites can be a problem if researchers cannot weed through the information to find what is important in a reasonable amount of time, as Matthias Mann of the Max Planck Institute of Biochemistry (Germany) explains. "Now we're in the paradoxical situation that we almost have too much of a good thing. So it's now possible to quantify even 10,000 phosphorylation sites in a single experiment. But in my view ... without then knowing which ones of the 10,000 are actually relevant for [a particular] biological system, then it's too much," he says.With this flood of new information about protein phosphorylation, the intricacy of the cell signaling network is becoming more apparent. "In the past with proteomics, people thought, 'Okay, one gene, one functionsone gene, one protein,'" says Albert Heck of Utrecht University (The Netherlands). "It makes you wonder and makes you humble about what we know already." Researchers are learning that the cell signaling pathways are incredibly complicated. "We think we understand sometimes how proteins are interacting with each other and how signaling networks are really working," says Emanuel Petricoin of George Mason University. "We go into these clinical trials where we think we can turn off these pathways, and you have some compensatory feedback loop that was unknown take over and actually turn on another pathway that you didn't even know was connected." Elucidating the structure and function of these ...

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A survey of computational methods and error rate estimation procedures for peptide and protein identification in shotgun proteomics

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