# MALDI-TOF/MS Analysis of Extracellular Vesicles Released by Cancer Cells
## 2 Applications
### Brain Tumors  
Brain tumors can be categorized into several kinds. Among them, the most diffused and lethal primary brain cancers are the heterogeneous group of gliomas, which can be classified according to their histological features, molecular specifications, location, differentiation patterns, and anaplasia features [50]. Through tissues arising from biopsy or tumor resection, the World Health Organization discriminates between low- (stage I and II with a relatively good prognosis) and high-grade (stage III and IV, or glioblastoma) tumors. Noninvasive approaches for the diagnosis of glioma tumors are highly needed, and EVs could play a key role in their diagnosis, prognosis, and follow-up, as demonstrated by several existing applications [50]. Useful information was also obtained by MALDI analysis of exosomes.  
[PERSON] et al. selected three different cell lines, human embryonic kidney 293, human H4 glioma, and mouse glioma Tu-2449, for a MALDI-based study dedicated to the characterization of N-glycans from EV glycoproteins [51]. Specifically, EVs secreted by the cells were isolated using ultracentrifugation and compared with total cellular membranes. Peptide N glycosidase F was used to release N-Glycans, which were labeled with 2-aminobenzzamide and analyzed by MALDI after an LC separation step. In all the cell lines, complex N-glycans (high amounts) were observed only in EVs, whereas high-mannose glycans were present in both EVs (small amounts) and cellular membranes (high amounts). In addition, some glycans specific to human and mouse cell lines have also been identified. The approach could represent a valuable help for the individuation of new glioma biomarkers and could further aid the understanding of the functions of glycans in EVs. Glioblastoma, the highest-grade form of glioma as well as one of the most aggressive types among all cancers, exhibits a high cell proliferation rate and infiltrating capacity of the surrounding tissues, which are related to the presence of cancer stem cells, small populations of tumor cells similar to stem cells that are characterized by stem cell marker expression, high self-renewal, and resistance to radiation/chemotherapy agents. In a study developed by [PERSON] et al. [52], EVs released in vitro by cancer stem-like cells obtained from two patients with primary glioblastoma were isolated by sequential centrifugal ultrafiltration and divided into two subtypes by electron microscopy and Western blot analysis. Then, 2-DE followed by MALDI-TOF analysis permitted the identification of specific proteins with different functions for each EV subpopulation, which could represent novel biomarkers or potential drug targets. However, a greater number of samples need to be analyzed to draw definitive conclusions. The major role played by EVs in pathological conditions was also understood by [PERSON] and co-workers, who described methods for the production, isolation, and characterization of the protein content of human cancer cell-derived exosomes in vitro [53]. Specifically, MALDI-TOF coupled to two-dimensional electrophoresis (2-DE) was exploited to obtain more detailed information on the proteome of exosomes secreted by brain tumor cells.