The application of mass spectrometry to identify disease biomarkers in clinical fluids like serum using high throughput protein expression profiling continues to evolve as technology development, clinical study design, and bioinformatics improve. Previous protein expression profiling studies have offered needed insight into issues of technical reproducibility, instrument calibration, sample preparation, study design, and supervised bioinformatic data analysis. In this overview, new strategies to increase the utility of protein expression profiling for clinical biomarker assay development are discussed with an emphasis on utilizing differential lectin-based glycoprotein capture and targeted immunoassays. The carbohydrate binding specificities of different lectins offer a biological affinity approach that complements existing mass spectrometer capabilities and retains automated throughput options. Specific examples using serum samples from prostate cancer and hepatocellular carcinoma subjects are provided along with suggested experimental strategies for integration of lectin-based methods into clinical fluid expression profiling strategies. Our example workflow incorporates the necessity of early validation in biomarker discovery using an immunoaffinity-based targeted analytical approach that integrates well with upstream discovery technologies.
Proteomic profiling of serum is an emerging technique to identify new biomarkers indicative of disease severity and progression. The objective of our study was to assess the use of surfaceenhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify multiple serum protein biomarkers for detection of liver disease progression to hepatocellular carcinoma (HCC). A cohort of 170 serum samples obtained from subjects in the United States with no liver disease (n ؍ 39), liver diseases not associated with cirrhosis (n ؍ 36), cirrhosis (n ؍ 38), or HCC (n ؍ 57) were applied to metal affinity protein chips for protein profiling by SELDI-TOF MS. Across the four test groups, 38 differentially expressed proteins were used to generate multiple decision classification trees to distinguish the known disease states. Analysis of a subset of samples with only hepatitis C virus (HCV)-related disease was emphasized. The serum protein profiles of control patients were readily distinguished from each HCV-associated disease state. Two-way comparisons of chronic hepatitis C, HCV cirrhosis, or HCV-HCC versus healthy had a sensitivity/specificity range of 74% to 95%. For distinguishing chronic HCV from HCV-HCC, a sensitivity of 61% and a specificity of 76% were obtained. However, when the values of known serum markers ␣ fetoprotein, des-gamma carboxyprothrombin, and GP73 were combined with the SELDI peak values, the sensitivity and specifity improved to 75% and 92%, respectively. In conclusion, SELDI-TOF MS serum profiling is able to distinguish HCC from liver disease before cirrhosis as well as cirrhosis, especially in patients with HCV infection compared with other etiologies. (HEPATOLOGY 2005;41:634-642.) T he incidence of hepatocellular carcinoma (HCC) continues to increase in the United States, 1 while, unfortunately, patient survival with HCC has only marginally improved over the last 20 years. Between 1981 and 1998, the 5-year survival rate only rose from 2% to 5%. 2 The poor survival rate is in part related to the diagnosis of HCC at advanced stages, where effective therapies are lacking. 3 Surveillance of patients at the highest risk for developing HCC (i.e., patients with cirrhosis) is an important strategy that can potentially decrease the cancer-related mortality rate. Although HCC meets the criteria of a tumor that would benefit from a surveillance program, the poor sensitivity and specificity of currently available tools has prevented widespread implementation of HCC surveillance. For example, ␣ fetoprotein (AFP) has been the serum marker that is most widely used for diagnosis as well as surveillance of HCC. 4,5 However, AFP levels may be normal in up to 40% of patients with HCC, particularly during the early stages (low sensitivity). 6 Furthermore, elevated AFP levels may be seen in patients with cirrhosis or exacerbations of chronic hepatitis (low specificity). 7 Prospective studies evaluating the performance characteristics of AFP for HCC surveillance
The increasing threat of bioterrorism and continued emergence of new infectious diseases has driven a major resurgence in biomedical research efforts to develop improved treatments, diagnostics and vaccines, as well as increase the fundamental understanding of the host immune response to infectious agents. The availability of multiple mass spectrometry platforms combined with multidimensional separation technologies and microbial genomic databases provides an unprecedented opportunity to develop these much needed resources. An overview of current proteomic strategies applied to microbes and viruses considered potential bioterrorism agents is presented. The emerging area of immunoproteomics as applied to the development of new vaccine targets is also summarized. These powerful research approaches can generate a multitude of potential new protein targets; however, translating these proteomic discoveries to useful counter-bioterrorism products will require large collaborative research efforts across multiple basic science and clinical disciplines. A translational proteomic research paradigm illustrating this approach using influenza virus as an example is discussed.
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