# MALDI-TOF/MS Analysis of Extracellular Vesicles Released by Cancer Cells
## 2 Applications
### Lung Cancer  
Lung cancer is the most frequent cancer worldwide and the major cause of death among all types [36]. It presents different histological subtypes, namely the most common non-small-cell lung cancer (  
new diagnostic strategies for the early diagnosis of these pathologies are crucial for proper management, improving the efficacy of treatments and survival rates of patients.  
Prostasomes are membranous microvesicles secreted by the epithelial cells of the prostate gland to deliver important information to sperm cells in semen, both in normal and in prostate cancer cells. A proteomic study on prostaglandomes from vertebral metastases of prostate cancer was conducted by [PERSON] et al. [45] using 2D-E coupled to MALDI-TOF. The presence among the proteins identified in the cancer-derived prostaglandomes of annexins (A1, A3, and A5) and dimethylarginine dimethylaminohydrolase 1, angiogenic factors that can favor tumor vascularization, supported the idea that prostaglandomes are involved in the interaction between tumor cells and their environment. Three complementary approaches based on MALDI, normal-phase liquid chromatography, and triple quadrupole MS, respectively, were used in a further application to obtain \(N\)-linked glycan profiles of expressed prostatic secretions and exosomes arising from normal individuals and high- and low-grade prostate cancer patients [46], finding changes in glycosylation that appeared to reflect the clinical status of prostate cancer.  
Exosomes were extracted from HT1376 bladder cancer cell lines using sucrose cushion ultracentrifugation and the analysis of the relevant proteome was performed by LC-MALDI-TOF/TOF [47]. High-quality identification was achieved for 353 exosomal proteins (72 for the first time), finding strong correlations between the observed proteome and carcinoma of the bladder and other sites using the ExoCarta and Gene Ontology databases. Preliminary data relevant to the possible presence of candidate markers in urinary exosomes from bladder cancer patients were also reported. In another work, urinary exosomes were purified by ultracentrifugation from the urine of patients affected by urothelial carcinoma, the predominant histologic type of bladder cancer, and normal subjects prior to the MALDI-TOF analysis, with the aim of detecting new reliable biomarkers for the pathology [48]. Using optimized isolation and storage protocols, specific m/z ions (3367, 3441, 3483, and 10,884) were individuated as exosome markers, whereas the m/z ions 5593 and 5947, which were identified as fragmented peptides of alpha-1-antitrypsin and histone H2B1K, respectively, were proposed as biomarkers for urothelial carcinoma. The discovery of these markers, also verified by immunohistochemical analysis, could represent a valid aid for the rapid diagnosis and prediction of the risks for relapse and progression. The potential of exosomes as a source of cancer biomarkers was also explored by [PERSON] et al. in a study focusing on characterizing their glycoconjugate content for future uses of compounds identified as biomarkers for genitourinary tract diseases [49]. Vesicles were isolated from normal urine samples using multistep differential centrifugation. Then, MALDI and LC/MS-MS analyses were independently performed on the extracts obtained through different pre-treatment steps, permitting the detection and identification of paucimmunosidic, high-mannose, and complex-type glycans.