# MALDI-TOF/MS Analysis of Extracellular Vesicles Released by Cancer Cells
###### Abstract  
The direct shedding of extracellular vesicles (EVs) from the plasma membrane is a recognized fundamental method for the intercellular transfer of properties in both physiological and pathological conditions. EVs are classified according to origin, biogenesis, size, content, surface markers, and/or functional properties, and contain various bioactive molecules depending on the physiological state and the type of the cells of origin including lipids, nucleic acids, and proteins. The presence of tumor-derived EVs in body fluids such as blood, ascites, urine, and saliva, together with the important role played in the tumor microenvironment where they intervene at different levels from oncogenesis to metastasis, make EVs a priority target for cancer studies. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can play a leading role in the analysis and characterization of EVs and their load due to its intrinsic advantages such as high throughput, low sample consumption, speed, the cost-effectiveness of the analysis, and the ease of use. This work reviews the main MALDI-TOF applications for the analysis and characterization of extracellular vesicles in the tumor field.  
MALDI-TOF MS; cancer; extracellular vesicles; exosomes; diagnosis +
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systems between cell proliferation and death [8], leading to the transformation of healthy cells into malignant ones. During the process, cancer cells must actively communicate with neighboring cells and the tumor microenvironment [9; 10], where EVs are considered key components in cell-to-cell mediation and intervene at different levels from oncogenesis to metastasis. Therefore, their potential use for cancer therapy and vaccines or drug delivery systems is being seriously considered [11; 12]. Furthermore, the abundant presence of tumor-derived EVs in biological fluids, together with the variability of their internal composition related to the physiological state, makes them interesting clinical targets to be used as biomarkers for the diagnosis and prognosis of diseases, carrying tumor-specific molecular signatures [13; 14].  
However, the characterization of extracellular vesicles and their load is not an easy task due to a series of issues, such as the standardization of isolation procedures, obtaining good quality extracts, long analysis time, high costs, the need for technical training, and many others [15], that make their use difficult in clinical practice. It has therefore become clear that reliable methods for a rapid and cost-effective analysis of EVs are highly advisable.  
A leading role in the field of complex samples analysis is certainly played by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), a technique that has clearly demonstrated its potential in providing information from complex biological samples for diagnostic purposes in a relatively simple way [16], particularly when it comes to cancer [17]. In fact, MALDI is characterized by high throughput, low sample consumption, speed, and cost-effectiveness of the analysis, as well as ease of use even by unskilled personnel, allowing the fast generation of spectra and/ or profiles from the matrix under study. Specifically, the potential, disadvantages, and possible implementation strategies of MALDI analysis of exosomes have recently been thoroughly discussed [18].  
The purpose of the present work is to comprehensively review the main existing literature relating to MALDI-TOF applications to the analysis and characterization of extracellular vesicles in the tumor field.