MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that post-transcriptionally regulate gene expression, and play key roles in the regulation of a variety of cellular processes and in disease. New tools to analyze miRNAs will add understanding of the physiological origins and biological functions of this class of molecules. In this study we investigate the utility of high resolution mass spectrometry for the analysis of miRNAs through proof-of-concept experiments. We demonstrate the ability of mass spectrometry to resolve and separate miRNAs and corresponding 3′ variants in mixtures. The mass accuracy of the monoisotopic deprotonated peaks from various miRNAs is in the low ppm range. We compare fragmentation of miRNA by collision-induced dissociation (CID) and by higher-energy collisional dissociation (HCD) which yields similar sequence coverage from both methods but additional fragmentation by HCD versus CID. We measure the linear dynamic range, limit of detection, and limit of quantitation of miRNA loaded onto a C18 column. Lastly we explore the use of data dependent acquisition of MS/MS spectra of miRNA during online LC-MS and demonstrate that multiple charge states can be fragmented, yielding nearly full sequence coverage of miRNA on a chromatographic time scale. We conclude that high resolution mass spectrometry allows the separation and measurement of miRNAs in mixtures and a standard LC-MS setup can be adapted for online analysis of these molecules.

The human genome encodes 11 cysteine cathepsins belonging to the papain-like family of cysteine peptidases that are known predominantly as endo-lysosomal enzymes. However, it is now understood that the functions and activities of cysteine cathepsins are not limited to endo-lysosomal compartments, as they are also active in the peri-and extracellular space. The thyroid gland is an endocrine organ where such intra-and extracellular proteolytic activities are required to solubilize the prohormone thyroglobulin from its luminal, covalently cross-linked storage forms for subsequent processing into smaller protein fragments and thyroid hormone liberation. Cathepsin K has been identified as one of the cysteine cathepsins with a crucial role in thyroglobulin processing. However, cathepsin K has mainly been a key focus of attention in the last few years because of its high expression in osteoclasts and due to its essential role as collagenase and elastase important for bone remodelling. Besides its remarkable function as an endopeptidase acting on highmolecular mass, covalently cross-linked extracellular substrates such as type I collagen, elastin or thyroglobulin, cathepsin K is also one of the very few proteolytic enzymes that is able to directly liberate thyroxine from thyroglobulin fragments by exopeptidase action. Thus, thyroid cathepsin K is now accepted as a cysteine peptidase with a vital role in liberation of thyroid hormones, which in turn are essential for homoeostasis by triggering a number of important biological processes, ranging from growth and brain development in young vertebrates to tissue remodelling events during morphogenesis or wound healing, as well as control of metabolic pathways and thermoregulation in adults. This review focuses on thyroid cathepsin K and will discuss how localization and trafficking within thyroid epithelial cells explain its thyroid-specific functions. The effects of targeted cathepsin K gene ablation will be summarized from the perspective of the thyroid gland, and we will propose potential consequences of short-and long-term inhibition of thyroid cathepsin K activity for the main thyroid hormone target tissues, namely bone, cardiovascular and immune systems, intestine, and the central nervous system, in addition to the thyroid gland itself.

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Cysteine cathepsins are expressed in most tissues, including the gastrointestinal tract. We demonstrated an involvement of mouse intestinal cathepsin B in extracellular matrix remodeling for regeneration from trauma. The present study aimed at elucidating roles of cysteine cathepsins in the non-traumatized gastrointestinal tract of mice. Thus we investigated expression and localization patterns of cathepsin B and its closest relative, cathepsin X, along the length of the gastrointestinal tract, and determined the effects of their absence. Cathepsin B showed the highest protein levels in the anterior segments of the gastrointestinal tract, whereas the highest activity was observed in the jejunum, as revealed by cathepsin B-specific activity-based probe labeling. Cathepsin X was most abundant in the jejunum and protein levels were elevated in duodenum and colon of Ctsb-/- mice. The segmental pattern of cathepsin expression was reflected by a compartmentalized distribution of junction proteins and basal lamina constituents, changes in tissue architecture and altered activities of the brush border enzyme aminopeptidase N. In conclusion, we observed different compensatory effects and activity levels of cysteine peptidases along the length of the small and large intestines in a segment-specific manner suggesting specific in situ functions of these enzymes in particular parts of the gastrointestinal tract.

Cathepsin K has been shown to exhibit antimicrobial and anti-inflammatory activities in the mouse colon. To further elucidate its role, we used Ctsk−/− mice and demonstrated that the absence of cathepsin K was accompanied by elevated protein levels of related cysteine cathepsins (cathepsins B, L, and X) in the colon. In principle, such changes could result in altered subcellular localization; however, the trafficking of cysteine cathepsins was not affected in the colon of Ctsk−/− mice. However, cathepsin K deficiency affected the extracellular matrix constituents, as higher amounts of collagen IV and laminin were observed. Moreover, the localization pattern of the intercellular junction proteins E-cadherin and occludin was altered in the colon of Ctsk−/− mice, suggesting potential impairment of the barrier function. Thus, we used an ex vivo method for assessing the mucus layers and showed that the absence of cathepsin K had no influence on mucus organization and growth. The data of this study support the notion that cathepsin K contributes to intestinal homeostasis and tissue architecture, but the lack of cathepsin K activity is not expected to affect the mucus-depending barrier functions of the mouse colon. These results are important with regard to oral administration of cathepsin K inhibitors that are currently under investigation in clinical trials.

Cathepsin B has been shown to not only reside within endo-lysosomes of intestinal epithelial cells, but it was also secreted into the extracellular space of intestinal mucosa in physiological and pathological conditions. In an effort to further investigate the function of this protease in the intestine, we generated a transgenic mouse model that would enable us to visualize the localization of cathepsin B in vivo. Previously we showed that the A33-antigen promoter could be successfully used in vitro in order to express cathepsin B-green fluorescent protein chimeras in cells that co-expressed the intestine-specific transcription factor Cdx1. In this study an analog approach was used to express chimeric cathepsin B specifically in the intestine of transgenic animals. No overt phenotype was observed for the transgenic mice that reproduced normally. Biochemical and morphological studies confirmed that the overall intestinal phenotype including the structure and polarity of this tissue as well as cell numbers and differentiation states were not altered in the A33-CathB-EGFP mice when compared to wild type animals. However, transgenic expression of chimeric cathepsin B could not be visualized because it was not translated in situ although the transgene was maintained over several generations.

Glycosylation is one of the most ubiquitous protein post-translational modifications observed in eukaryotic organisms. In cancer, key cellular pathways and interactions, related to invasion and metastasis, are disrupted by altered protein glycosylation. Many current clinical cancer biomarkers are glycoproteins (i.e. CEA, CA15-5, CA125, and PSA). Glycosylation is known to be aberrant in breast cancer. However, systematic studies to identify glycoproteins in cancer have been limited, and typically focus on single proteins of interest or glycans released from proteins. We have developed a novel protein fractionation and enrichment strategy for glycoproteomic analysis of the secreted proteins, incorporating lectin affinity capture for glycoprotein enrichment, intact protein separation, and enzymatic digestion followed by LC-MS/MS analysis. Here, we apply this workflow to capture and analyze glycoproteins in the cancer secretome, which includes proteins secreted and/or shed from cancer cells into the extracellular space. Four breast cancer cell lines (HCC70, HCC1806, BT474, and ZR75-1) and one normal mammary epithelial cell line (MCF10A) were subjected to glycoproteomic analysis. Proteins corresponding to 3360 unique gene names were identified. Subsequent data analysis yielded a variety of types of information about glycoproteins in the cell line secretomes. 1) Fucosylated and branched-sugar containing glycoforms of proteins were identified by lectin binding properties. For example, PHA-P/PHA-L lectins showed high affinity for aldolase, immunoglobulins, and inter-alpha-trypsin inhibitor, suggesting these proteins contain branched sugar structures. 2) Enrichment of some proteins was observed in breast cancer cell lines compared to the normal mammary epithelial cell line and vice versa. For example, high expression of fucosylated vimentin was observed in MCF10A, but not breast cancer cell lines. 3) Differences in glycosylation patterns of proteins were observed between cell lines. For example, the protease legumain was observed to be highly fucosylated in MCF10A, but had a higher affinity for PHA-P/PHA-L in HCC70. Additional substrates and inhibitors of legumain were also identified. Proteins of interest were further studied in additional follow-up experiments including western blotting and glycostaining. Additional proteins of interest were measured in plasma samples from women with (benign or malignant) breast lesions. Citation Format: Maria Arampatzidou, Majlinda Kullolli, Sharon J. Pitteri. Glycoproteomic analysis of breast cancer cell lines for biomarker discovery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2489. doi:10.1158/1538-7445.AM2014-2489