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Gene expression assay guides chem otherapy For millions of women diagnosed with breast cancer, the trialand-error process of finding effective drugs for post-operative chemotherapy is often unpredictable and unpleasant. However, Jenny Chang and colleagues at the Baylor College of Medicine believe they have developed a pre-operative method to reduce the guesswork involved. Using a microarray, the researchers identify gene expression patterns in breast cancer specimens before the start of chemotherapy to determine if a patient will respond positively to the use of docetaxel, one of the most effective postoperative breast cancer drugs. The researchers took biopsy samples from 24 patients who were subsequently treated with four 3-week-long cycles of docetaxel. Tumor sizes were measured after each cycle, and the patients were ultimately classified as resistant (>25% residual disease; 13 patients) or sensitive (≤25% residual disease; 11 patients) to docetaxel treatment.
Attom olardetection of proteins Chad Mirkin and colleagues at Northwestern University have developed a new ultrasensitive method for protein detection. The researchers can detect prostate-specific antigen (PSA) at 30 aM. Adding a PCR step to the protocol drives down the limit of detection to 3 aM, which the researchers say is 6 orders of magnitude more sensitive than commercial assays.
Ultrasensitive detection.Magnetic microparticle–prostate-specific antigen–gold nanoparticle sandwich. (Adapted with permission. Copyright 2003 American Association for the Advancement of Science.)
The researchers use magnetic microparticles (MMPs) with bound monoclonal PSA antibodies in addition to gold © 2003 American Chemical Society
nanoparticles (NPs) with bound polyclonal PSA antibodies and hybridized oligonucleotides, which are called bar codes. When the modified MMPs and NPs are added to a sample, both types of antibodies bind to PSA molecules, forming a sandwich. To remove the MMP–PSA–NP complexes, Mirkin and colleagues apply a magnetic field to the reaction container. Washing the complexes dehybridizes the bar codes, which are left behind after another application of a magnetic field. The residual singlestranded bar codes are analyzed in one of two ways. The researchers devised a PCRless method, in which the bar code is sandwiched between a chip-bound oligo that is complementary to half of the bar code sequence and a NPbound oligo that is complementary to the other half of the sequence. Mirkin and colleagues perform a “scanometric” assay in which the sandwich is exposed to a silver amplification solution and imaged on a flatbed scanner. This allowed the researchers to quantitate the amount of
Gene expression analysis of the biopsy samples yielded 1628 genes that had significant variance when resistant and sensitive tumors were compared; these genes were considered informative. Statistical analysis revealed that 92 of those genes showed 2.6–4.2-fold decreases or 2.5–15.7-fold increases in resistant versus sensitive tumors and thus might predict the efficacy of docetaxel. The researchers’ findings suggest that patterns of expression for sensitive and resistant breast cancer tumors involve multiple gene pathways and that the integration of these genes is more likely to predict the success of particular drug treatments than single-gene biomarkers. They also noted that resistant tumors often overexpressed genes associated with protein translation, cell cycle, and RNA transcription, whereas sensitive tumors overexpressed genes involved in stressor apoptosis, protein transport, and RNA splicing, among other areas. (The Lancet 2003, 362, 362–369)
recovered bar code DNA. Alternatively, PCR can be performed on the bar code DNA prior to scanometric detection to increase sensitivity. (Science 2003, 301, 1884– 1886)
M aking m alaria less m ysterious
profile P. falciparum and tentatively assign cellular functions to the thousands of previously uncharacterized genes. The array contained 367,226 probes, for an average of 1 probe for every 150 bases on both DNA strands. Of the 5159 predicted genes on the array, 4457 (88%) were expressed in at least one of the nine life-cycle stages examined. Of those, 43% were regulated by the cell cycle, while 51% were constitutively expressed. Expression levels covered 5 orders of magnitude.
Previously published work with Plasmodium falciparum—the parasite that causes the most severe form of malaria, which kills 1.5–2.7 million children in sub-Saharan Africa per year—has shown that at least 2391 of its genes are coded into proteins. However, until now, the functions for 65% of those genes have been unknown. Elizabeth Winzeler and Karine Le Roche at The Scripps Research Institute and their colleagues at the Genomics Institute of the Novartis Research Foundation, the Naval Medical Research Center, the National Institute for Ring oftranscription.Expression levels and proportion of genes (outer ring) at Medical Research (U.K.), various stages in the transcription process. and Walter Reed Army (Adapted with permission. Copyright 2003 Hospital are using a cusAmerican Association for the Advancement tom-made, high-density of Science.) oligonucleotide array to JournalofProteom e Research •Vol. 2, No. 6, 2003
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currents One goal of the experiment was to show that genes with similar functions have similar expression profiles, which would allow unknown genes to be rapidly and empirically assigned functions. The genes were grouped on the basis of time of expression, and cluster analyses were performed. Looking at the known genes within the clusters confirmed that genes with highly correlated expression levels and similar patterns of expression over time were often involved in similar functions or cellular processes. Winzeler, Le Roche, and colleagues believe that assigning cellular functions to the thousands of uncharacterized proteins will provide detailed descriptions of gene expression throughout the P. falciparum life cycle and will make it possible to create vaccines that are more protein-specific in their targets and more effective. (Science 2003, 301, 1503–1508)
Lipidom ics by M S Ian Blair and co-workers at the University of Pennsylvania School of Medicine and the Vanderbilt University Medical Center use an electron capture atmospheric pressure chemical ionization (APCI) MS/MS method to analyze isomers. The researchers say that the method is 2 orders of magnitude more sensitive than conventional negativeion APCI-MS. The researchers add a pentafluorobenzyl (PFB) moiety to linoleic acid, arachidonic acid, and their derivatives prior to MS analysis. PFB captures electrons produced in the APCI-MS process and dissociates, resulting in a PFB radical and a negatively charged lipid molecule. Although Blair and coworkers could distinguish between various lipids using electron capture dissociation APCI-MS/MS alone, they were not able to resolve isomeric
Clinicalproteom ics:Are w e there yet? Frustrated by a limited ability to detect cancer and other diseases in their earliest stages, some researchers are turning to clinical proteomics to find differences in the patterns of proteins expressed in diseased versus healthy states. Advocates say that clinical proteomics has the potential to save patients from unnecessary biopsies and organ removal, but other experts argue that it is not ready for real-world application. Katie Cottingham explores both sides of the matter in an Analytical Chemistry feature article. At the heart of the controversy is the method that several clinical proteomics researchers are using: SELDI MS, which is similar to MALDI, except the surfaces of SELDI protein chips contain chromatographic arrays. Samples are run from patients with a disease (typically cancer) and from healthy patients, and bioinformatics software is used to determine the discriminating patterns of peaks. Another question that is strongly debated is whether clinical proteomics researchers can rely on such patterns of peaks or whether they ought to identify some of the peaks. There are also concerns that telltale proteins might not be present at detectable levels in the early stages of disease, but newer data shows that highly abundant carrier proteins are sticking to the SELDI surface, bringing the less abundant biomarkers with them. Many issues, such as cost, false positives, and reproducibility, must be resolved before SELDI MS will be widely embraced by the medical community, the article concludes. But if clinical proteomics strategies stand up to the scrutiny, the rewards could be tremendous. (Anal. Chem. 2003, 75, 472 A–476 A)
currents species. To separate isomers, the researchers ran the PFBtagged lipids on a chiral-phase LC column with a methanol/ isopropanol/hexane solvent prior to multiple reaction monitoring (MRM) MS. The chiral LC/electron capture APCI-MRM-MS protocol was also used to identify lipid metabolites in rat cells. The researchers monitored the levels of the metabolites when the cells were treated with an antioxidant and observed a large increase in the levels of two types of prostaglandin, PGE2 and PGE2␣. PGE2 levels rose from 0.14 to 0.70 nM, and PGE2␣ levels rose from
