Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis.

Owing to its cross-linking effects, it is currently believed that formalin fixation of routinely processed tissues in the clinic prevents protein extraction and profiling. The aim of our study was to develop a robust, fast, standardized, and easy to use technique for the solubilization of non-degraded, full length, and immunoreactive proteins from formalin-fixed tissues for western blot and protein microarray analysis. Sections of routinely processed formalin-fixed and paraffin-embedded tissues of various origin were analysed. After deparaffination, tissues were manually dissected from the slides and transferred into an optimized protein extraction buffer system. Proteins were solubilized and subsequently analysed by western blot and reverse phase protein microarrays. We succeeded in isolating non-degraded, soluble, and immunoreactive proteins from routinely processed formalin-fixed tissues. We were able to detect membrane, cytoplasmic and nuclear proteins at the expected molecular weight. No differences were found in the protein yield and protein abundances between fresh frozen and formalin-fixed tissues. Using western blots and reverse phase protein microarrays, the receptor tyrosine kinase HER2, an important protein target for antibody based cancer treatment, was reliably measured in formalin-fixed breast cancer biopsy samples when compared with measurement by immunohistochemistry and fluorescence in situ hybridization; remarkably, immunohistochemically equivocal cases (score 2+) can be categorized according to HER2 protein abundance. Our new clinically orientated multiplexed protein measurement system may be generally applicable to determine the relative abundances of known disease-related proteins in small amounts of routinely processed formalin-fixed tissue samples for research and diagnosis. This technique may also be used to identify, characterize, and validate known and new protein markers in a variety of human diseases.

Clinical proteomics: new trends for protein microarrays.

Protein microarrays are an emerging class of nanotechnology for tracking many different proteins simultaneously. Much progress has been made for applications in basic sciences. Translation of these methods for the treatment of patients, however, is slow, because the realities in the clinic are rarely taken into account, and proteomic changes in cultured cell lines might not fully reflect human diseases due to the lack of the tissue microenvironment. In this review, we summarise current protein microarray approaches that are being developed for profiling tissues and histopathologically defined cell populations from cancer patients. We provide an overview of clinical applications for protein lysate microarrays and discuss the power of this technology for the discovery of disease markers for cancer diagnosis and individualised treatment.

Responses of rumen microflora to high-concentrate low-roughage diets containing whey products.

After 3 wk on a standardization ration of alfalfa hay and corn silage ad libitum and concentrate at 1 kg/3 kg milk, 15 lactating Holstein cows were fed 2.3 kg hay/day and one of five concentrate rations ad libitum for a 6-wk experiment. Cows were returned to the standardization ration after the experimental period. Concentrate rations during experimental period were: 1) control, 2) 14% dried whole whey, 3) 5.9% high mineral whey product, 4) 11.8% demineralized whey product, and 5) 9.8% lactose. Ration 3 contained the same amount of minerals from whey as ration 2 while rations 4 and 5 contained the same amounts of lactose as ration 2. Whey products replaced portions of corn and soybean meal in the rations. Total protozoa numbers in rumen contents averaged 1.8 X 105 /ml and were not different among times although they tended to be less during the experimental period on ration 4. Dasytricba decreased on rations 1, 2, and 4, while trends in numbers of Isotricha, Entodinium, and Diplodinium were not consistent. Only a few Ophryscolex were in a couple of the rumen samples. Bacterial numbers increased from 4.0 X 109/ml during standardization to 5.8 X 109/ml during the high-grain period, then returned to 3.8 X 109/ml in the post-experimental period. The number of lactose fermenters increased on all diets containing whey or whey products but not on the control diet. No differences in numbers of starch digesters were detected between times or among experimental rations because of large variations in numbers. Numbers of proteolytic organisms were not different among experimental rations but were generally lower during the post-experimental period.