Ovarian cancer is a fatal gynaecological malignancy in adult women with a five-year overall survival rate of only 30%. Glycomic and glycoproteomic profiling studies have reported extensive protein glycosylation pattern alterations in ovarian cancer. Therefore, spatio-temporal investigation of these glycosylation changes may unearth tissue-specific changes that occur in the development and progression of ovarian cancer. A novel method for investigating tissue-specific N-linked glycans is using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) on formalin-fixed paraffinembedded ( Ovarian cancer is the fifth most fatal malignancy in adult women with an estimated 21,290 new cases diagnosed and 14,180 deaths recorded in the United States during 2015 (1). There are several reasons for the poor prognosis of ovarian cancer and its diagnosis at advanced stage-lack of diagnostic markers for the early detection (2, 3), rapid metastasis of the disease (4), and limited or modest understanding of the etiology, origin and the diverse clinical and pathological behavior of the tumors (5). Moreover, epithelial ovarian cancer comprises of several distinct sub-types based on their histopathological features into serous, endometrioid, clear-cell, mucinous, and undifferentiated subtypes (6, 7).Protein glycosylation is an important post-translational modification which has relevance in many biological processes such as cell signaling, immune responses, extracellular interaction and cell adhesion (8, 9). Aberrant protein glycosylation such as the expression of truncated glycans as well From the ‡Faculty

Amyloid fibrils are aggregated and precipitated forms of protein in which the protein exists in highly ordered, long, unbranching threadlike formations that are stable and resistant to degradation by proteases. Fibril formation is an ordered process that typically involves the unfolding of a protein to partially folded states that subsequently interact and aggregate through a nucleation-dependent mechanism. Here we report on studies investigating the molecular basis of the inherent propensity of the milk protein, -casein, to form amyloid fibrils. Using reduced and carboxymethylated -casein (RCM-CN), we show that fibril formation is accompanied by a characteristic increase in thioflavin T fluorescence intensity, solution turbidity, and ␤-sheet content of the protein. However, the lag phase of RCM-CN fibril formation is independent of protein concentration, and the rate of fibril formation does not increase upon the addition of seeds (preformed fibrils). Therefore, its mechanism of fibril formation differs from the archetypal nucleationdependent aggregation mechanism. By digestion with trypsin or proteinase K and identification by mass spectrometry, we have determined that the region from Tyr 25 to Lys 86 is incorporated into the core of the fibrils. We suggest that this region, which is predicted to be aggregation-prone, accounts for the amyloidogenic nature of -casein. Based on these data, we propose that fibril formation by RCM-CN occurs through a novel mechanism whereby the rate-limiting step is the dissociation of an amyloidogenic precursor from an oligomeric state rather than the formation of stable nuclei, as has been described for most other fibril-forming systems.

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Imaging mass spectrometry (IMS) is a powerful technology for mapping distributions of biological molecules like proteins and peptides within tissue sections. It is therefore potentially extremely useful for the analysis of pathological conditions such as neoplastic diseases. The use of IMS is typically limited to fresh frozen tissue specimens. However, there is a high interest in the possibility of being able to analyze the tissue proteome of formalin-fixed paraffin-embedded (FFPE) specimens that have been stored together with the clinicopathological information of patients in huge archives over many decades. We have therefore developed an antigen-retrieval protocol using a high temperature citric acid buffer to allow partial reversal of FFPE protein cross-linking. Coupled with automated deposition of trypsin and matrix, our method allows the generation of meaningful peptide ion distribution images. In situ peptide fragmentation provided identification of high abundance proteins such as Actin and Collagen. Furthermore, downstream application of three different HPLC-MS strategies allowed identification of a maximum of 106 proteins, 67 of which were mass correlated to ions from IMS analysis of archived FFPE ovarian tissue. The CAAR method presented here complements previously described antigen-retrieval protocols and is an important step in being able to fully analyze the proteome of archived FFPE tissue.

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