High-sensitivity blood-based detection of breast cancer by multi photon detection diagnostic proteomics.

We have developed several new methods for blood-based cancer detection by diagnostic proteomics. Ultrasensitive methods of immunoassay using multiphoton-detection (IA/MPD) increase sensitivity by 200- to 1,000-fold (1 femtogram/mL). This has allowed the measurement of cancer biomarkers with very low concentrations in blood that could not be measured for full patient cohorts with conventional immunoassays. Sensitivity and specificity in cancer detection have been found to be potentiated by use of immunoassay panels which include tissue-specific cancer biomarkers as well as cytokines and angiogenic factors. The ultrasensitive immunoassays revealed that patient to patient variations in the concentrations of individual biomarkers in blood can extend over many orders of magnitude (up to six) and that the distributions of biomarker concentrations over patient cohorts are non-Gaussian. New methods of data analysis which correlate abundances of multiple, different biomarkers have been developed to deal with such data sets. Sensitivity and specificity of about 95% have been achieved for blood-based detection of breast cancer in pilot studies on 250 patients and 95 controls. Pilot studies indicate that this methodology may also allow differentiation of malignant breast cancer from benign lesions and can provide similar sensitivity and specificity for other epithelial cancers such as prostate cancer, ovarian cancer and melanoma. The methods developed for selection, application, and evaluation of very high sensitivity biomarker panels are expected to have general relevance for diagnostic proteomics.

Looking for Thom's biomarkers with proteomics.

In recent years, large numbers of putative disease biomarkers have been identified. Combinations of protein biomarkers have been proposed to overcome the lack of single, magic-bullet identifiers of disease conditions. The number of biomarkers in a panel must be kept small to avoid the combinatorial explosion that requires very large, uneconomical sample cohorts for validation. Recent results on high sensitivity blood-based diagnostic proteomics (Godovac-Zimmermann, J et al., J. Proteome Res. 2006) suggest that the keys to identifying useful panels include judicious application of physiological knowledge to choose appropriate combinations of local, tissue/disease markers and global, systemic markers and to use very high sensitivity protein detection. Biomarkers that show non-Gaussian landscapes reminiscent of Rene Thom's multiple, stable-state landscapes seem to have the greatest predictive value for breast cancer (Godovac-Zimmermann, J. et al., J. Proteome Res. 2006).

Ultra-sensitive immunoassays using multi-photon-detection in diagnostic proteomics of blood.

Multiphoton-detection methods that detect as little as 1000 atoms of (125)I-streptavidin increase the sensitivity of immunoassays by 200- to 1000-fold (1 femtogram/mL). Improved background suppression allows 20- to 100-fold improvements in sensitivity for conventional immunoassays (10-50 femtogram/mL). Quantitation of low abundance biomarkers in blood (PSA, TNFalpha, VEGF, IL-1beta, IL-6, and IL-8), for the first time for complete patient cohorts, indicates that very high analytical sensitivity and new statistical methods are crucial for serum-based diagnostic proteomics.

Ultra-high sensitivity multi-photon detection imaging in proteomics analyses.

We report on the use of 125I and 131I labeling and of new, multicolor, multi-photon detection (MPD) methods to routinely and quantitatively detect protein spots on two-dimensional gel electrophoresis plates in the zeptomole to attomole range. We demonstrate that the MPD methodology can be used to detect radioactive labels on two-dimensional gels and has several characteristics that are advantageous for functional proteomics. First, by using single particle detectors, the sensitivity for detection of radiolabels can be improved dramatically. Second, because single particle detectors can differentiate the particle energies produced by different decay processes, it is possible to choose combinations of radioisotopes that can be detected and quantified individually on the same 2-D gel. Third, the MPD technology is essentially linear over six to seven orders of magnitude, i.e., it is possible to accurately quantify radiolabeled proteins over a range from at least 60 zeptomoles to 60 femtomoles. Finally for radionuclides that decay by electron capture, e.g., with emission of both beta and gamma rays, co-incident detection of two particles/photons can be used to detect such radionuclides well below background radiation levels. These methods are used to monitor acidic/phosphorylated proteins in as little as 60 ng of HeLa cells proteins.

Perspectives in spicing up proteomics with splicing.

In the post-genomics era there has been an acceleration of understanding of cellular and organismal biology and this acceleration has moved the goalposts for proteomics. Higher eukaryotes use alternative promoters, alternative splicing, RNA editing and post-translational modification to produce multiple isoforms of proteins from single genes. Switching amongst these isoforms is a major mechanism for control of cellular function. At present fundamental limitations in sensitivity, in absolute quantitation of proteins and in the characterization of protein structure at functionally important levels strongly limit the applicability of proteomics to higher eukaryotes. Recent developments suggest that quantitative, top-down proteomics analyses of complete proteins at sub-attomole levels are necessary for physiologically relevant studies of higher eukaryotes. New proteomics technologies which will ensure the future of proteomics as an important technology in medicine and cellular biology of higher eukaryotes are becoming available.