SCIENCE & TECHNOLOGY
CHEMICAL APPROACH Technique replaces phosphoserine (shown) and phosphothreonine side chains with aminoethylcysteine to make those phosphorylated residues sensitive to cleavage by trypsinlike enzymes.
CHEMICAL PROTEOMICS Chemistry-based technique maps phosphorylation sites in proteins
and professor of biochemistry and molecular biology Matthias Mann of the University of Southern Denmark. But with t h e LC/MS/MS approach, Shokat says, "the site of phosphorylation is often not possible to detect—especially if it is a long peptide or if the residue modified is distant from either end of the peptide." In addition, "the actual signal detected for negatively charged phosphoserine is quite weak." T h e chemistry-based approach sidesteps these problems. But its reported use has so far been restricted to mapping of serine and threonine phosphorylation in purified proteins and relatively simple protein mixtures, whereas LC/MS/MS has been used to map serine, threonine, and tyrosine phosphorylation in thousands of proteins from whole-cell lysates [Nat. Biotechnol, 2 0 , 3 0 1 (2002)}. Associate professor of chemistry Benjamin F. Cravatt I I I of Scripps Research Institute calls the chemistry-based technique "quite exciting" and says it "should gain general utility." However, how well will it work "for identifying phosphorylation sites on proteins from complex proteomes?" he asks. "The next step is clearly to apply this strategy to cell and tissue
phorylation in eukaryotic cells," Knight the human body are phos- says. Phosphates are first removed from phorylated after they are bio- phosphorylated serine and threonine synthesized. Now, a chem- residues and replaced with new side chains istry-based strategy has been that resemble lysine—target residues for developed to map nearly all phosphory- protein cleavage by trypsinlike enzymes. lation sites in proteins. After enzymatic cleavage of The technique was devised by graduate the modified protein, the student Zachary A. Knight and professor resulting peptide fragments of cellular and molecular pharmacology are analyzed by mass spectrometry (MS) to deterKevan M. Shokat of the University of California, San Francisco, and coworkers [Nat. mine the original phosphoBiotechnol., published online Aug. 17, rylation pattern. The mass http://dx.doi.org/10.1038/nbt863}. Knight of the lysine mimic differs conceived the approach and earned agrand from that of lysine, preprize for it (shared with Shokat) in the Na- venting normal lysine tional Inventors Hall of Fame 2002 Col- residues from being mischaracterized as phospholegiate Inventors Competition. Phosphorylation plays a key role in reg- rylation sites. ulating the production and transport of In addition to serine and hormones and nutrients, and aberrant threonine, tyrosine residues Knight Shokat phosphorylation is involved in diseases are also phosphorylated in ~~ ~~ ranging from cancer to diabetes. In fact, a small but functionally significant minor- extracts and see what one can discover." "nearly every cellular process is controlled ity ofphosphoproteins. "While our chemShokat's group is indeed now trying to at some level by protein phosphorylation," istry is not applicable to phosphotyrosine, make the technique useful for larger sets of Knight says. The ability to map phospho- in unpublished work we have investigated proteins. T h e challenge is to make the rylation sites in proteins is therefore an im- engineering a protease so the phosphospe- chemical conversions "scalable to the proportant research goal. cific proteolysis approach can be extend- teomic scale," Shokat says. W h a t is needed are "transformations that are robust, efIn the new method, phosphorylated ed to phosphotyrosine," Knight says. residues are converted to aminoethylcysT h e chemistry-based technique will ficient, and scalable to abundant as well as teine amino acids, which can then be se- complement the current technique for low-copy-number proteins." lectively cleaved by trypsinlike proteases. phosphorylation mapping—the use of liqUltimately, he and his coworkers also "This is like the discovery of the first re- uid chromatography and t a n d e m M S hope to extend the technique to other striction enzyme," Shokat says—but with (LC/MS/MS) to sequence tryptic phos- modifications, such as glycosylation. "We specificity for phosphorylated amino acids phopeptides. "That method has been re- envision a rapid expansion ofenzyme tools instead of a particular DNA sequence. fined over the years and is now quite good," for selective interrogation of the proteome, The technique detects phosphorylation Shokat says. Leading researchers in the with tailor-made sequence specificity for at serine and threonine sites, which repre- field, he says, are chemistry professor Don- all natural post-translational modificasent "more than 99% of protein phos- ald F. Hunt at the University of Virginia tions," they note.—STU B0RMAN
O
NE-THIRD OF ALL PROTEINS IN
HTTP://WWW.CEN-ONLINE.ORG
C&EN
/ SEPTEMBER
1. 2003
31
