Serum Protein Profiling
Serum protein profiling has a number of advantages, including convenience for the patient and allowing for frequent screening of patients—especially younger women or those with hereditary predisposition. In addition, protein patterns in serum reflect pathologic changes and specific disease-related changes result in different protein expression, noted Rob Tollenaar, Ph.D., professor of surgical oncology at Leiden University Medical Center. He presented work on proteomic expression done in concert with Dr. Tempst’s lab.
This methodology has not been without its challenges. “Sample handling is problematic,” he said, particularly when there are differences in how samples are handled, whether they are left too long at room temperature, and what sort of tubes are used—“all these things can compromise your data.”
The objective of Dr. Tollenaar’s study was to assess the feasibility of a mass spec approach for the detection of breast cancer. Using a randomized block design, pre-operative serum samples obtained from 78 breast cancer patients and 29 controls were used to generate high-resolution MALDI-TOF protein profiles.
“The spectra generated using C8 magnetic beads assisted mass spec were smoothed, binned, and normalized after baseline correction. Preliminary data suggest that the high sensitivity and specificity indicate the potential usefulness of serum protein profiles for the detection of breast cancer,” said Dr. Tollenaar. “This method and technology are ready for the analysis of a larger patient series.”
The themes of discovery and validation resonated throughout the conference. Dr. Schiess presented a two-stage strategy for discovery and initial validation of serum biomarkers corresponding to specific cancer-causing mutations that involved the use of mass spec assisted discovery, verification, and validation of disease biomarkers. “I take a systems approach and study the mechanisms of the disease by using a mouse model to study tumors with silent progression,” he said. “I used prostate cancer as a model, but I believe applications can be made to other cancers as well.”
Dr. Schiess integrated his study of mouse genetics with proteomics techniques for the diagnosis and stratification of patients with prostate cancer. “We identified a set of biomarkers predictive for the genetic status in human prostate cancer patients, thus identifying potential responders to cancer therapies targeting specific pathways.”
In the initial discovery phase, Dr. Schiess and his group detected and identified N-linked glycoproteins with distinguishable expression patterns in primary normal and diseased tissue. “The proteins identified in the initial phase will be subjected to targeted MS analysis in plasma samples using highly sensitive and specific selected reaction-monitoring technology. Since glycosylated proteins, such as those secreted or shed from the cell surface, are likely to reside and persist in blood, the two-stage strategy is focused on the quantification of tissue-derived glycoproteins in plasma.”
Dr. Schiess noted that the focus on N-glycoproteome not only reduces the complexity of the analytes, but also targets an information-rich subproteome—relevant for remote sensing of diseases in the plasma. “The discovery and validation workflow allows for the robust identification of protein candidate panels that can be selectively monitored in blood plasma at high sensitivity in a reliable, noninvasive, and quantitative fashion.
“We further discovered serum biomarkers for the prognosis of localized prostate cancer providing additional noninvasive prognostic markers for cancer aggressiveness, and thereby, supporting the decision of active surveillance or immediate surgical intervention.”