Comprehensive glyco-proteomics analysis of human plasma from patients with hereditary breast cancer using a multi-lectin affinity column approach

Protein glycosylation, one of the commonest post-translational modifications, is known to play a major role in a diverse set of biological processes, such as the immune response and regulation of cellular homeostasis. It has been shown that glycosylation is involved in signaling pathways associated with tumorigenesis. Germ-line mutations in BRCA1/2 genes account for most of hereditary breast and ovarian cancer (BC, BOC). Gene expression profiling studies have disclosed a specific molecular signature for BRCA1/2 breast tumors as compared to sporadic cases. In this study, a multi-lectin-affinity column approach, coupled with immunodepletion of high abundance proteins, was used to investigate tumor-specific changes in the plasma glyco-proteome of BC patients and healthy family members sharing the same BRCA1 gene founder mutation (5083del19), previously reported by our group, with the aim to identify potentially specific biomarkers. Plasma samples were collected according with the HUPO-PPP guidelines, aliquoted and stored at 80 C. To evaluate changes in the glycosylation pattern, a 2D gel electrophoresis (GE) coupled with mass spectrometry (MS) was used. Approximately 50 protein spots were detected by 2D-GE. Proteins, whose expression was significantly different among the two groups (patients vs healthy carriers), were excised, trypsin-digested and analyzed by MALDI-TOF. The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. We identified 8 unique glycoproteins that were differentially expressed in the two populations. Moreover, some of them showed a different pattern of glycosylation, suggesting that quantitative and qualitative changes are present in the glyco-proteome of affected BC individuals compared to their healthy relatives, even though both are sharing the same genetic alteration. The results of this study appear very promising and may contribute to shed more light on the molecular mechanisms underlying tumorigenesis in a specific (BRCA1-positive) subset of BC patients.