M E E T I N G Rajendrani Mukhopadhyay reports from Pittcon 2004— Chicago, Ill.
time,” says Mendrinos. Because of the low sensitivity, pathologists tend to report negative results to clinicians. This either delays proper treatment or forces clinicians to undertake invasive procedures for diagnosis. SELDI-TOFMS provides better sensitivity than examinations of the fluid. “We’re able to increase the sensitivity of detection from 30% for [the examination] in our series of samples to 90% sensitivity, and up to 85% specificity, by just using 100 µL of fluid with only one type of chemical surface,” says Mendrinos. Mendrinos and colleagues studied CSF samples from 30 patients and discovered protein fingerprints that could distinguish between cancer and noncancerous diseases such as multiple sclerosis. Based on the limited data, it also seemed that the protein fingerprints within the cancer cases are different enough to distinguish between primary and metastatic tumors. Likewise, Bowser and his colleagues found unique biomarkers for ALS, a
fatal disease in which motor neurons die, leaving the victim paralyzed but mentally alert. There are no diagnostic markers for the disease and clinical diagnoses can take up to a year. “It seemed like a perfect opportunity to apply [MS-based] proteomics to come up, hopefully, with a better way to diagnose the disease more rapidly,” says Bowser. He and his colleagues studied the CSF of 25 ALS patients and 35 control subjects and found that accurate diagnoses, based on the distinctive proteomic fingerprint of ALS, could be made within several hours. Bowser and colleagues are trying to identify the 10 biomarkers found in their study. They are also interested in tracking changes in expression of the biomarkers as the disease advances. Bowser explains, “[If we know] how the biomarker pattern changes as the disease progresses, we can then use it in clinical trials to test drugs.” Both Mendrinos’ and Bowser’s groups are now conducting their studies with larger groups of patients.
PAUL CREMER, TEXAS A&M UNIVERSITY
The researchers used this system to demonstrate that the binding of the 2,4DNP antibody to its ligand is bivalent— there are two distinct binding events. Measurements were sensitive enough for Cremer and his co-workers to calculate the binding constants for the two individual binding events (J. Am. Chem. Soc. 2003, 125, 4779– 4784). Now the researchers have started to investigate the effect cholesterol has on the binding between the 2,4-DNP antibody and its ligand. Cholesterol is an abundant molecule in eukaryotic cell membranes—one cholesterol molecule can be present for every phospholipid molecule. Although cholesterol is known to make a lipid membrane less fluid, its effects on protein interactions
within lipid membranes is not well established. Cremer’s group discovered that cholesterol affects the bivalent binding of the 2,4-DNP antibody. The first binding event of the antibody to 2,4-DNP is enhanced by cholesterol, but the second binding event is actually inhibited. “This result was the big surprise at the end of the day,” says Cremer. The researchers’ discovery demonstrates how cholesterol, on its own in a simple phospholipid membrane, can significantly modify receptor–ligand interactions. As Cremer sums it up, “In this particular case, cholesterol is an inhibitor of multivalency.” Cholesterol’s effect on the binding events of the 2,4-DNP antibody cannot be attributed to lipid rafts, which are thought to modulate protein interactions in lipid membranes. Cremer says he and his colleagues went out of their way to make sure lipid rafts were not formed in their experimental system. Cremer emphasizes that cholesterol’s inhibitory effect on multivalent receptor–ligand interactions has so far been shown only for his particular model system with 2,4-DNP. It still remains to be seen whether cholesterol’s effect on protein binding is exerted on other receptor–ligand pairs in different lipid systems.
Cholesterol affects protein interactions in lipid membranes Numerous biological processes, including signal transduction, cell motility, and vesicle fusion, depend on interactions between proteins and their respective ligands. In some cases, the ligand, the protein receptor, or both are confined to a lipid membrane. So the question arises whether the surrounding lipid molecules have an impact on the receptor–ligand interactions. According to Paul Cremer and his research group at Texas A&M University, the answer is yes. Cremer and his co-workers have been studying proteins in phospholipid membranes for several years. They have designed a high-throughput microfluidics device that consists of microchannels coated with a phospholipid bilayer that is modified with 2,4-dinitrophenyl (2,4-DNP) ligands. A fluorescently labeled antibody that recognizes 2,4DNP is introduced into the microchannels, and the binding of the antibody to 2,4-DNP is monitored by total internal reflection fluorescence microscopy.
Rajendrani Mukhopadhyay reports from Experimental Biology 2004—Washington, D.C. CNS proteomics Two groups presented proteomic analyses of neurological diseases at the Experimental Biology meeting in April. Savvas Mendrinos and colleagues at Emory University identified protein biomarkers for cancerous and non-cancerous diseases of the central nervous system (CNS). Robert Bowser and his colleagues at the University of Pittsburgh focused on amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease) and found 10 unique biomarkers associated with the disease. Both groups used SELDI-TOFMS to analyze cerebrospinal fluid (CSF). Because the CSF bathes the brain and spinal cord, pathologists usually examine the cells sloughed off from the tissues into the fluid to determine CNS disease states. However, “The sensitivity of the examination is less than 30% most of the
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Cholesterol modifies binding. The presence of cholesterol in the phospholipid bilayer (pink) can modify the binding of the 2,4-DNP antibody (green) to 2,4-DNP (yellow).
Journal of Proteome Research • Vol. 3, No. 3, 2004
