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.

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.

Slow step in activation of muscle glycogen phosphorylase b by adenosine 5'-monophosphate.

The incubation of glycogen phosphorylase b from rabbit skeletal muscles with the allosteric activator (AMP) for 10-15 min has been found to result in an additional increase in the initial rate of the enzymatic reaction (v) as compared with the corresponding value of v measured by the initiation of the enzymatic reaction by the addition of a mixture of glucose 1-phosphate and AMP (0.02 M HEPES, pH 6.8; 37 degrees C).

Slow conformational transitions of muscle glycogen phosphorylase b induced by specific ligands.

The effect of specific ligands (the substrate glucose 1-phosphate, AMP, and flavins) on the initial rate of tryptic digestion of muscle glycogen phosphorylase b has been studied (0.02 M HEPES, pH 6.8; 37 degrees C).

Effect of flavins on the rate of proteolytic digestion of muscle glycogen phosphorylase b.

The kinetics of tryptic proteolysis of rabbit skeletal muscle phosphorylase b has been registered by the diminishing of protein fluorescence intensity at lambda = 335 nm (excitation at 290 nm) or by the disappearance of the enzyme activity (0.02 M Hepes buffer, pH 6.8, 37 degrees C).