Abstract
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.
Highlights
Building from what was learned in the first wave of serum protein expression profiling studies, strategies striking a balance of sample throughput with improved depth of serum protein capture and protein identification need to continue to evolve
Accommodating these concerns into proteomic analysis design is facilitated by use of the lectins as front-end fractionation and enrichment tools
We have only described the use of six lectins, and there are dozens of other individual lectin types available commercially that remain to be empirically assessed and hundreds more described in the literature [21, 45]
Summary
It has long been known that cellular glycosylation profiles change significantly during oncogenesis (18 –20); the search continues for tumor-secreted glycoproteins that can serve as biomarkers for diagnostics and/or tumor markers of the biological changes associated with the altered glycosyla-. ␣-Fetoprotein (AFP) is another well characterized serum glycoprotein used as a surrogate marker of the presence of liver cancers, and multiple glycoforms of AFP have been identified (33, 39 – 41) Specific targeting of these PSA and AFP glycoforms as the diagnosis for disease state or similar characterization of other known serum components like prostate-specific membrane antigen represents a targeted proteomic strategy amenable to quantitative mass spec-. Trometry strategies that utilize isotope tagging or mass shift labeling techniques [42, 43]
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