Meeting News
Katie Cottingham reports from the HUPO Sixth Annual World Congress—Seoul
biomarkers. This liver study, however, is the first one in which the researchers have taken the next step and looked for candidate markers in the blood. Microscope slides containing slices of biopsied liver tissue from seven patients who developed cirrhosis as a result of hepatitis C infection were obtained. For each tissue slice, proteins from a healthy section and a diseased section were compared. One challenge facing the researchers, however, was the limited amount of starting material (1000–3000 cells). Therefore, they separated the proteins with a modified 2D difference gel electrophoresis (DIGE)
vestigators currently are collaborating with Qi-Hong Sun at SKD Biotech, Ltd. (China), and Fuchu He at the Beijing Proteome Research Center and the BeiDiscovering biomarkers with jing Institute of Radiation Medicine to high-performance proteomics produce antibodies against the rest of According to Helmut Meyer at Ruhr the candidates. University Bochum (Germany), Surprisingly, two of the proteins that many proteomics researchers today passed the validation steps (b-tropoare not conducting studies on the myosin and transgelin) typically are basis of sound scientific principles. found in muscle, not liver. When the For example, experiments are being researchers looked up these proteins in performed only once, and samples the literature, they read that liver steloften are pooled to eliminate or reduce late cells are transformed myogenically biological variability. In reaction to upon hepatitis C infection. “Although this, he advocates “high-performance it was known in the literature, nobody proteomics”. With this so far had ever looked at paradigm, proteomics exwhether those muscle properiments are quantitative teins might show up in the and are conducted many serum,” says Meyer. He extimes. Biological differences plains that because the liver Patients are not averaged away; inis the source of many blood MS Microdissection 2DE stead, practitioners of highproteins, it makes sense performance proteomics that b-tropomyosin and (+) (–) Serumanalyze biological replicates transgelin eventually wound marker candidate independently. Results from up in serum. These proteins, ELISA such experiments tend to be however, would not have Western blot reproducible and meaningbeen detected directly in seful, says Meyer. rum samples by MS because Protein ID High-performance prothey are present in very low Blood teomics is a general concept concentrations, he adds. Serum/plasma IHC validation that can be applied to any Meyer says the team still set of proteomics experihas a lot of work to do. For ments, and Meyer and colexample, in the current Searching in tissues. High-performance proteomics workflow for the leagues have applied it most study, healthy and diseased discovery of biomarkers in tissue. After candidate biomarkers are idenrecently to the search for sections of a tissue slice tified from tissue samples, the researchers look for these markers in secirrhosis biomarkers. With from each patient were rum samples. (Figure adapted with permission from Proteomics 2007, 7 [S1], 18–26. Copyright 2007 Wiley-VCH Verlag GmbH & Co.) this guiding principle, they compared. In the future, the have discovered that >30 researchers plan to comproteins are differentially pare tissue samples from regulated in damaged liver cells comprotocol. “We are doing full-saturation healthy control subjects and cirrhosis pared with healthy ones in cirrhosis 2D DIGE, which is ~100-fold more senpatients. In addition, they will colpatients. Furthermore, at least two of sitive than the high-sensitivity silver laborate with physicians to obtain more these potential biomarkers also are stain,” explains Meyer. blood samples collected with their present in the serum of patients. After the differentially expressed pronewly developed standard operating A blood-based test is the ultimate teins were identified by MS, validation procedures. The remaining candidate goal, but Meyer says that the notion studies were conducted. The expression proteins will be run through the validaof starting a biomarker search by exlevels of the candidate marker proteins tion process after antibodies against amining blood samples is “nonsense”, were assessed with immunohistochemthese proteins are generated. “We will because the types of proteins that are istry (IHC) techniques. Finally, the also apply the methodology from the expected to be good biomarkers are researchers performed western blots beginning for other liver diseases, such present in the blood at concentrations on patient sera samples. Although >30 as liver cancer,” says Meyer. Finally, that are too low for current instruments proteins were differentially expressed, the researchers are collaborating with to detect. So, he and his co-workers use antibodies against only 3 of these were Bruker and Protagen to adapt the high-performance proteomics on tissue commercially available for validation high-performance proteomics method samples to find candidate biomarkers. purposes. “We discovered, and other for LC/MS workflows and to develop Then, they look for these proteins in colleagues are discovering, that the operational software. “The nice thing is the blood with targeted assays. Previnumber of accessible antibodies is very that the LC/MS procedure we have deously, they have examined pancreatic limited,” says Meyer. “So the validation veloped is ~10-fold more sensitive than cancer and kidney tissues for possible has really slowed down now.” The inthe 2D DIGE” version, he says.
4546 Journal of Proteome Research • Vol. 6, No. 12, 2007
These days, more and more proteomics researchers are touting diseased tissue as the ideal source for biomarker discovery. In this strategy, blood samples are analyzed only at a later stage for the presence of potential markers. These scientists point out that the proteins in blood products such as plasma or serum have too great a dynamic range for current instrumentation to handle and that interesting proteins are present at concentrations that are too low to detect without targeted assays. However, Sam Hanash at the Fred Hutchinson Cancer Research Center says that reports of the demise of blood as a biomarker source are greatly exaggerated. As evidence of this, he points to several candidate biomarkers that he and colleagues at various institutions recently have identified for many cancers. Working with blood is challenging, but so is working with tissue or other biological fluids, Hanash explains. For example, a tumor is composed of many cell types that may differ from those in a normal organ. Thus, it is not obvious which tissue should be used as the control. “You may be comparing apples with oranges as opposed to comparing diseased apples with normal apples,” he says. In addition, he cites examples of known biomarkers such as PSA that are not overexpressed in diseased tissue but are present at higher levels in the blood of cancer patients than in the blood of controls. Such markers wouldn’t be picked up in tissue proteomics investigations. With these studies, “you are at a loss when you generate all these data to be able
New version of Human Protein Atlas
Mathias Uhlén of the Royal Institute of Technology (Sweden) announced the latest version of the Human Protein Atlas (www.proteinatlas.org), a resource that provides immunohistochemical staining profiles for various proteins. With version 3.0, visitors to the site can access the validation data for the 3000 antibodies used. Also, new confocal fluorescence microscopy images help researchers determine the subcellular localizations of queried proteins.
to pinpoint which ones are candidate markers to [search for] in the blood,” he says. With extensive fractionation, or “decomplexing”, of blood samples, Hanash and other consortia members funded by the National Cancer Institute’s Early Detection Research Network and various foundations have discovered SAM HANASH
Biomarker discovery with blood samples
In the blood. Autoantibodies in the serum of a cancer patient bind to tumor proteins spotted on a microarray slide. Red spots indicate intense reactivity.
possible markers for breast, lung, pancreatic, and colon cancers. As an example, he points to the lung-cancer study. Lysates from lung tumors and lung-cancer cell lines were fractionated with ion-exchange and reversed-phase LC, and then the fractions were spotted onto microarrays that were exposed to plasma from patients with lung cancer. Autoantibodies to several proteins were
Finally, an advanced search function allows more flexible queries than was previously possible.
Protein standards still being developed
At the Protein Standards Initiative session, advances were announced for two major efforts. Alex Bell of McGill University (Canada) reported that only 6 of 24 laboratories identified all 20 proteins in the HUPO standard developed by Invitrogen. He says that some researchers didn’t load enough proteins,
identified with this method. “The immune system is recognizing the presence of aberrant proteins and is making autoantibodies against them early in tumor development,” explains Hanash. The researchers also detected circulating biomarkers with other methods. These candidates have undergone a successful round of blinded validation on independent samples, he notes. Another important aspect of the cancer studies was the types of samples that were analyzed. Hanash says that the disease specimens were well matched to the controls to avoid biases. In addition, the consortia had access to preclinical samples, which were taken from groups of subjects before any of the participants were diagnosed with a disease. Such specimens are valuable for the discovery of early disease indicators, and they are free of collection biases. “Those samples were really challenging to obtain; they were just not available until recently,” he says. Now, researchers who run large clinical studies are more aware of potential sample-collection biases. Also, some older studies already included such specimens, which have been sitting in freezers. He explains that, in some cases, scientists had to be convinced that the technology had progressed to a point where useful information could be gleaned from these specimens. Although Hanash has had successes with blood samples, he isn’t advising anyone to drop their studies on tissues or biological fluids other than blood. In fact, he sees a bright side to this situation. “If the rest of the world wants to work with tissues, then we won’t have any competition!” he proclaims.
had digestion problems, or were too stringent with database searches. With assistance, most of the labs eventually detected all of the proteins. Invitrogen is scaling up the production of the standard. Jeff Turner of Sigma-Aldrich described the Universal Proteomics Standard 2 (UPS2), which includes 48 proteins at levels that span a dynamic range of 5 orders of magnitude. The four outside labs that tested the standard detected 17–27 of the proteins. UPS2 is projected to be commercially available by the end of 2007.
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