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Methodologies

Lectin Capture Strategies Combined with Mass Spectrometry for the Discovery of Serum Glycoprotein Biomarkers^*

Richard R. Drake, E. Ellen Schwegler, Gunjan Malik, Jose Diaz, Timothy Block, Anand Mehta and O. John Semmes^,¶

From the Center for Biomedical Proteomics, Virginia Prostate Center, and Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia 23507 and Drexel Institute of Biotechnology and Virology Research, Doylestown, Pennsylvania 18901

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.

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