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R E S E A R C H 16 O/ 18 O
labeling in the spotlight
To quantitate changes in protein expression between two samples, proteomics researchers routinely use stable isotope dilution techniques, such as metabolic labeling or isotope-coded affinity tagging (ICAT). But as two research groups report in this issue of the Journal of Proteome Research, the 16O/18O isotopic labeling method is gaining in popularity. Since the late 1990s, Catherine Fenselau and her colleagues at the University of Maryland, College Park, have been adapting 16O/18O labeling to quantitative proteomics studies. She says that 16O/18O labeling is unique among isotope dilution strategies because it is catalyzed by an enzymatic reaction. Fenselau explains that with ICAT, for instance, peptides are tagged via a chemical reaction, which generates byproducts that must be removed prior to analysis. Byproducts are avoided with the 16O/18O method, however, because two 16O atoms on the carboxyl termini of peptides are exchanged for two 18O atoms in a reaction catalyzed by trypsin. Barry Karger at Northeastern University agrees with Fenselau and adds, “16O/18O is a very nice way to go in terms of differential expression when you have, in this case, limited sample.” Whereas commercial ICAT kits recommend >100 µg of total protein, Karger says that in his collaborative research on breast cancer biopsies with colleagues at his university and at Massachusetts General Hospital, he has only 1–4 µg of total protein with which to work. In this issue of JPR, both Karger and Fenselau use the 16O/18O labeling method to investigate breast cancer, but each researcher’s group probes a different question. Whereas Karger and colleagues (pp 604–612) apply the technique in their hunt for diagnostic breast cancer biomarkers, Fenselau and Brown (pp 455–462) look for proteins indicative of whether a tumor will respond to a chemotherapeutic agent. Using laser-capture microdissection (LCM), Karger’s team can harvest about 10,000 cells from human biopsies at one
© 2004 American Chemical Society
P R O F I L E
time. The researchers obtained one set of cells from normal breast tissue and a second set of cells from breast cancer tissue. Cells were lysed and digested separately. In the next step, the peptides from normal cells were treated with H18 2 O. The other set of peptides was simultaneously manipulated in an identical manner, except that H16 2 O was used. The samples were combined in a 1:1 ratio, separated by reversed-phase LC, and analyzed by MS/MS on an ion trap instrument. 18O-labeled peptides are 4 Da heavier than their unlabeled counterparts, thus peptides from normal and metastatic cells can be easily compared on the same mass spectrum.
(a)
(b)
LCM-captured cells. (a) Normal and (b) carcinoma cells after LCM. (Adapted with permission. Copyright 2003 American Association for Clinical Chemistry.)
Seventy-six proteins were identified with this procedure, and some of these were differentially regulated in cancer cells. Isocitrate dehydrogenase, for example, was expressed 6.4-fold higher in metastatic cells than in normal cells. The mRNA for this protein is also highly expressed in breast cancer cells, as reported in earlier microarray studies.
Discovering previously identified biomarkers has led Karger to conclude that the method works quite well. “We think this type of approach will become a generic approach for looking at different types of tissues with LCM samples,” he says. Since the paper was submitted, Karger says that the team has dramatically improved the sample preparation process. They are also using a new ion trap-FTMS instrument for MS/MS analysis, which is yielding promising results. Fenselau and Brown used a very similar protocol, but there are important differences between the two studies. Instead of biopsies, they used cultured breast cancer cell lines. “We’re working with cell culture partly to find out what proteins should be looked at and then, eventually, we would want to move to clinical samples,” says Fenselau. Also, samples were fractionated at the protein level prior to trypsin digestion. The researchers did this to reduce the complexity and the dynamic range of the samples. “There are too many peptides for HPLC to separate well,” Fenselau points out. The researchers compared the protein levels in breast cancer cells that are either resistant or sensitive to the chemotherapy drug doxorubicin. After the LC and digestion steps, peptides from the doxorubicin-sensitive cells were labeled with 18O, and peptides from resistant cells were exposed to non-isotopic water. The labeled and unlabeled peptides were then mixed together and analyzed by MS/MS with a quadrupole-TOF instrument. Of the 22 proteins that were identified, several were significantly up-regulated in resistant cells, such as metallothionein, ubiquitin pathway proteins, and apoptosis inhibitors. Fenselau says that many of these have been identified in studies of other cancer cell lines. Although both groups say that much more work is necessary, it is already clear that 16O/18O labeling, in combination with LC/MS/MS, has the potential to be a valuable tool for biomarker discovery. —Katie Cottingham
Journal of Proteome Research • Vol. 3, No. 3, 2004
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