Novel comprehensive approach for accessible biomarker identification and absolute quantification from precious human tissues

The identification of specific biomarkers obtained directly from human pathological lesions remains a major challenge, because the amount of tissue available is often very limited. We have developed a novel, comprehensive, and efficient method permitting the identification and absolute quantification of potentially accessible proteins in such precious samples. This protein subclass comprises cell membrane associated and extracellular proteins, which are reachable by systemically deliverable substances and hence especially suitable for diagnosis and targeted therapy applications. To isolate such proteins, we exploited the ability of chemically modified biotin to label ex vivo accessible proteins and the fact that most of these proteins are glycosylated. This approach consists of three successive steps involving first the linkage of potentially accessible proteins to biotin molecules followed by their purification. The remaining proteins are then subjected to glycopeptide isolation. Finally, the analysis of the nonglycosylated peptides and their involvement in an in silico method increased the confident identification of glycoproteins. The value of the technique was demonstrated on human breast cancer tissue samples originating from 5 individuals. Altogether, the method delivered quantitative data on more than 400 potentially accessible proteins (per sample and replicate). In comparison to biotinylation or glycoprotein analysis alone, the sequential method significantly increased the number (≥30% and ≥50% respectively) of potentially therapeutically and diagnostically valuable proteins. The sequential method led to the identification of 93 differentially modulated proteins, among which several were not reported to be associated with the breast cancer. One of these novel potential biomarkers was CD276, a cell membrane-associated glycoprotein. The immunohistochemistry analysis showed that CD276 is significantly differentially expressed in a series of breast cancer lesions. Due to the fact that our technology is applicable to any type of tissue biopsy, it bears the ability to accelerate the discovery of new relevant biomarkers in a broad spectrum of pathologies.