Kinase Cartography

Courtesy of Zachary Knight BEAUTIFUL CLEAVAGE: Trypsin cleaving a hypothetical protein into smaller fragments with C-terminal phosphorylated residues--a basis for phosphorylation mapping techniques. Proteins communicate with each other through posttranslational modifications, and locating modified sites is a key challenge in proteomics. Phosphorylation, the most common modification by far, is central to cell signaling, and knowledge of where and when proteins are phosphorylated could help researchers decipher disease mechanisms, including those for cancer and diabetes. Because protein modifications are not genetically encoded, it's difficult to track phosphorylation, says chemistry professor Kevan Shokat, University of California, San Francisco. A new technique that swaps phosphorylation sites for other more easily identifiable chemical species may enable fast and widespread identification, aiding researchers in decoding the posttranslational language of proteins. Proteomicists praise the elegance of the procedure, and the work earned first author Zachary Knight a grand prize in the 2002 Collegiate Inventors Competition. A COURSE CHARTED The most commonly used method for mapping such sites is proteolytic digestion of the protein, followed by tandem mass spectrometry of the resulting fragments. This technique can fail, however, when used to analyze low-abundance proteins or proteins with substoichiometric phosphorylation (that is, not all of the molecules are phosphorylated). Moreover, no naturally occurring proteases cleave specifically at phosphorylation sites, making it impossible to identify those sites if the phosphate groups are lost during proteolysis or ion bombardment. To solve these problems, Shokat and coworkers created a method through which the phosphate groups themselves can be adapted to facilitate site-specific cleavage. "It sort of cuts right to the chase. It cuts right at the amino acid that is phosphorylated, and thereby gives you very precise information about where phosphates have been attached," Shokat says. The new technique transforms phosphoserine and phosphothreonine residues into the lysine analogs aminoethylcysteine and b-methylaminoethylcysteine, respectively.1 An identical chemistry, applied to peptides rather than whole proteins, was described previously in a paper by Gary Hathaway and coworkers at the California Institute of Technology.2 After sites are converted, addition of a lysine-specific protease leads to cleavage at lysine residues and the modified sites. "Instead of having to sequence every individual peptide in order to find the phosphorylation site, which can often be technically difficult, you can instead look at the mass of the fragments that you get from the tryptic digest, and that alone should be sufficient to tell you where the sites are," says Knight. Kai Johnsson of the Federal Polytechnic School of Lausanne, Switzerland, says that he was impressed by the reaction's efficiency and the fact that it can be performed on a solid-phase support to enrich for phosphorylated peptides. Thomas Conrads, associate director of the Mass Spectrometry Center at the National Cancer Institute, calls the method "provocative" in that it could ultimately allow researchers to tease out changes in the phosphorylation states of proteins. Conrads notes that, thus far, the technique has been used only on known, model proteins. He says he would like to see it used to compare proteins from diseased and healthy samples to "try to understand how the flux of protein phosphorylation changes, [and] how the characters of all these different signal transduction pathways change with cell state or disease state." OPEN WATERS The technique is limited to serine and threonine residues and cannot map phosphorylated tyrosine sites. Shokat and Knight indicate in the paper's conclusion that they are working to resolve this limitation by designing a phosphotyrosine-specific protease. Bradford Gibson, professor and director of chemistry, Buck Institute of Age Research, Novato, Calif., collaborated with Shokat's group to ensure that the chemistry was optimized for mass spectrometry. Gibson says that one significant "next step" would be to show that the technique can be used for proteins isolated from one- and two-dimensional gels. Mapping the more than 300 possible protein modifications will likely mean tapping into chemical methods similar to those used by Knight and Shokat. Gibson notes that very few proteomics scientists today are familiar with decades-old solution-phase protein chemistry that they might be able to exploit. Says Gibson: "The key thing is what subset of these chemical methods can be adapted to proteomics, and also what new chemistries can be brought into the field." Aileen Constans can be contacted at aconstans@the-scientist.com. References 1. Z.A. Knight et al., "Phosphospecific proteolysis for mapping sites of protein phosphoryl-ation," Nat Biotechnol, 21:1047-54, September 2003. 2. F. Rusnak et al., "Identification of phosphorylated and glycosylated sites in peptides by chemically targeted proteolysis," J Biomol Tech, 13:228-37, 2002. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Kinase Cartography

Interested in reading more?

Become a Member of

Courtesy of Zachary Knight
BEAUTIFUL CLEAVAGE: Trypsin cleaving a hypothetical protein into smaller fragments with C-terminal phosphorylated residues--a basis for phosphorylation mapping techniques.