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MS puts a number on enzyme activity A new strategy measures the absolute amount of kinase activity in the PI3/Akt signal transduction pathway.
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activity in the pathway in absolute units. carried out with tumor biopsies from t’s like PCR for kinases. Pedro Cutilmice with melanomas and samples from Matthias Mann of the Max Planck las, Bart Vanhaesebroeck, and colpatients with acute myeloid leukemia. Institute for Biochemistry (Germany) leagues at the Ludwig Institute for The detection of differences indicates says that the idea is clever. “There’s no Cancer Research, University College that the assay could help clinicians sort PCR equivalent in proteomics, but usLondon, and the Royal Free & Univerout which patients suffer from defects ing enzyme activity may be the closest sity College Medical School (all in the in the PI3/Akt pathU.K.) have developed a techway. “You can imagnique that amplifies the signal ine [that if] 10–50% generated by kinases—enzymes of cancers have upthat add phosphate groups to regulated PI3 kinase proteins—and applied it to an signaling, it means important cell signaling path0 0.033 0.067 0.33 0.67 the [remaining] way. (Proc. Natl. Sci. U.S.A. Amount of lysate (µg) 50–90% don’t. It’s 2006, 103, 8959–8964) important for cliniThe investigators focused cians to know which on the kinases in the phosphoLC/MS chromatograms show that increasing amounts of cancer cell lysate patient may respond inositide 3-kinase (PI3)/Akt produce more phosphorylated synthetic peptide. The gray and black lines to drugs” that target signal transduction pathway. correspond to the amounts of internal standard and phosphorylated synthis pathway, says The pathway frequently goes thetic peptide, respectively. (Adapted with permission. Copyright 2006 Cutillas. haywire in a broad spectrum of National Academy of Sciences, U.S.A.) But Mann woncancers: ~40% of colorectalders about the ability to properly rething,” he says. Ruedi Aebersold of the and ~30% of breast-cancer cases result produce the assay in different labs. ReInstitute of Molecular Systems Biology from such mistakes. Currently, patholosearchers “have to take a biopsy from of ETH Zurich points out that previous gists carry out immunohistochemical studies have demonstrated the quantifica- the patient, lyse the cells, put the pepassays on tissue biopsies to determine tide in, and see how it’s phosphorylatwhether the enzymes in the pathway are tion of enzyme activity by laser-induced ed. If one lab extracts the biopsy a bit properly regulated. It isn’t a very consis- fluorescence measurements and CE in a differently and lyses the cells a bit differcomplex sample. In this case, he says, tent test because different pathologists ently, that will show up as a difference “what is novel is that the investigators can interpret the results in various ways. in measured activity,” he says. A single A quantitative measure of enzyme ac- used the combination of MS, targeted lab may be able to repeat the assay contivity really was needed. Cutillas realized analysis, and measurement of enzyme sistently, but different labs may carry activity.” that “a way to amplify the signal of a out the assay in slightly different ways. The investigators aren’t entirely sure protein would be to use its intrinsic enThus, it would be difficult to make whether the synthetic peptide is only zymatic activity. . . . If each kinase molecule converts 1000 substrates into pro- recognized by the kinases in the PI3/Akt measurement comparisons among labs, he adds. pathway. Cutillas points out, “We have ducts per second, allowing the reaction The investigators are now tackling over 500 kinases in the human genome.” to occur for 60 s gets 60,000 product other signal transduction pathways that But the controls they ran seem to sugmolecules.” are relevant in oncology, such as the migest the peptide is specific for the pathSo, the investigators decided to use togen-activated kinase or the JAK pathway: Treatment with pervanadate, an a synthetic peptide that was predomiways. “If it works for one pathway, agonist of the pathway, increased the nantly recognized by the kinases in the there’s no reason it couldn’t work for amount of phosphorylated peptide, PI3/Akt pathway. A known concentraother pathways. It’s a question of findwhereas treatment with wortmannin, tion of the peptide was incubated with ing the right substrates,” says Cutillas. an inhibitor, decreased the amount of a cell lysate for ~10 min. The investiThe goal of the investigators now is to the phosphorylated peptide. gators then quantified the amount of develop a multiplexed, clinical assay that Cutillas, Vanhaesebroeck, and colphosphorylated peptide against an intercan quantify the activity of kinases that leagues demonstrated that differences nal standard by MALDI TOFMS, LC/ belong to several different pathways. a in activation of the PI3/Akt pathway MS, or LC/MS/MS. They correlated —Rajendrani Mukhopadhyay could be discerned when the assay was this number with the amount of kinase 5950
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