*4.2. MS Acquisition*

During MS analysis, data-dependent acquisition (DDA) are normally used. Recently, data-independent acquisitions (DIA), such as SWATH (sequential window acquisition of all theoretical fragment ion), MSE, and multiplexed MS/MS, are used in EV studies to satisfy di fferent purposes. Unlike DDA, DIA simultaneously fragments all precursor ions present in a wide isolation window. Braga-Lagache et al. analyzed MV proteins from plasma samples by both DDA and multiplexed DIA on a quadrupole orbitrap instrument. In each cycle of multiplexed DIA, data is usually acquired with one full MS scan followed by a series of MS2, such as ten MS2 scans. Each MS2 scan records all the fragment ions generated by precursor ions that are isolated from multiple di fferent isolation windows with a fixed *m*/*z* range, such as isolated from three randomly combined 10 *m*/*z* isolation windows. A targeted approach is used to analyze the DIA data by using spectral libraries from formerly acquired fragment spectra with exact mass and retention time of precursors. They found that a multiplexed DIA approach only consumed one third of the DDA acquisition time when data was extracted by a targeted approach. Their results suggested that multiplexed DIA was a valuable alternative to DDA [103]. Moon et al. and Chutipongtanate et al. also applied DIA to analyze the protein content of EVs [32,42]. In the study of Moon et al., crude exosomes prepared by sucrose density ultracentrifugation were digested in-gel and analyzed by MS<sup>E</sup> on a Waters Q-TOF mass spectrometer. In MSE, alternating low- and high-energy collision-induced dissociation are used. The low-energy scan is used to obtain precursor information, while the high-energy scan is to collect fragment ions. A total of 1877 urinary exosome proteins were identified from IgA nephropathy and thin basement membrane nephropathy patients [42]. Chutipongtanate et al. utilized SWATH to analyze urinary EV proteins. In SWATH, the mass range of interest is divided into several segments with a fixed *m*/*z* range, such as 25 *<sup>m</sup>*/*<sup>z</sup>*. Then, precursor ions within each segmen<sup>t</sup> are fragmented together until all the segments are analyzed. They achieve a label-free DIA quantitative analysis for EV and MV proteins with a curated spectral library of 1145 targets, suggesting their potential clinical use [32].

Quantitative MS based on label and label-free have been demonstrated to study various diseases, such as prostate cancer, asthenozoospermia and venous thrombosis [39,40,46,104]. Fujita et al. labeled the urinary EV proteins with isobaric tag for relative and absolute quantitation (iTRAQ). A total of 4710 proteins were identified by MS, including 3528 proteins quantified [39]. Lin et al. quantified seminal EV proteins with iTRAQ labeling and revealed 91 proteins with significant changes [40]. 2D-LC and tandem mass tag (TMT) were also used to quantitative analysis of EVs in HIV-infected alcohol drinkers and cigarette smokers through precipitation-based isolation [104]. Although stable isotope labeling by amino acids in cell culture (SILAC) cannot label EV proteins from human biofluids, a PROMIS-Quan method which based on SILAC quantification was developed in order to gain a comprehensive quantification for potential clinical EV protein analysis. In PROMIS-Quan, EV lysates were spiked with super-SILAC which was prepared from cell cultures and served as an internal standard. Then, the same set of super-SILAC mix was quantified relative to purified proteins of interest, with known absolute amounts. By this way, EV proteins can be quantified not only in large-scale but also retrospectively only relative to the same set of super-SILAC standard [29]. Quantitative MS is not only applied to the EV studies with the aim of biomarker discovery but also developed as an evaluation method to assess the EV isolation. Wang et al. established a multiple reaction monitoring (MRM) based method to assess the purity of EVs. MRM is often used for target quantitative analysis as a validation method for biomarkers reported in discovery MS analysis. They first generated 15N-labeled quantification concatamers (QconCATs) for a pattern of targeted EV proteins and abundant serum proteins (non-EV proteins or contaminants) as the internal standards for quantification of those proteins in MRM. QconCATs were artificial proteins composed of concatenated tryptic peptides from targeted proteins. The purity of EVs was then assessed by the quantitative results of the targeted EV proteins and abundant serum proteins in MRM [105]. They further expanded this method to separate EVs and lipoprotein particles by adding QconCAT for apolipoproteins into the previous MRM assay [106].
