Chapter 2
Clinical Proteomics for the Analysis of Circulating
Extracellular Vesicles
Maria N. Barrachina and A ´ ngel Garcı ´a
Abstract
In recent years, technical improvements in proteomics have allowed its rapid application for biomarker
discovery, new drug target identification, and the study of disease progression and drug resistance. The
clinical potential of circulating extracellular vesicles (EVs) as a source of biomarkers is one of the reasons
why several research groups have recently applied proteomics to their study. A large variety of proteomic
approaches such as gel-based proteomics and bottom-up and top-down mass spectrometry have been
applied to the study of EVs. In this chapter, we will present basic protocols for gel-based and quantitative
MS-based approaches applied to the study of EVs.
Key words Extracellular vesicles, Platelets, 2D-DIGE, Label-free LC-MS/MS
1 Introduction
Over the past decade, the interest in extracellular vesicles (EVs) as a
novel source of biomarkers has increased dramatically due to their
implication in cell-to-cell communication, cellular activation,
inflammation, hemostasis, and disease development and progression [1]. EVs are a heterogeneous population of cell-derived circulating vesicles. However, there is some controversy on
nomenclature and sizes of the different types of vesicles. For this
reason, the International Society for Extracellular Vesicles (ISEV)
has provided some criteria to classify EVs, categorizing them into
three groups: microvesicles (MVs), exosomes, and apoptotic bodies
[2]. MVs (100–1000 nm) are formed by outward blebbing of the
plasma membrane and subsequent fission of plasma membrane
blebs. In contrast, exosomes (30–100 nm) are formed by multivesicular bodies being stored on the cytoplasm upon stimulus. The
multivesicular bodies fusion with the plasma membrane and release
their content (exosomes) to the extracellular environment
[3, 4]. Lastly, apoptotic bodies (500–4000 nm) are formed as a
result of the induction of cellular apoptosis [5].
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
13
Clinical Proteomics for the Analysis of Circulating
Extracellular Vesicles
Maria N. Barrachina and A ´ ngel Garcı ´a
Abstract
In recent years, technical improvements in proteomics have allowed its rapid application for biomarker
discovery, new drug target identification, and the study of disease progression and drug resistance. The
clinical potential of circulating extracellular vesicles (EVs) as a source of biomarkers is one of the reasons
why several research groups have recently applied proteomics to their study. A large variety of proteomic
approaches such as gel-based proteomics and bottom-up and top-down mass spectrometry have been
applied to the study of EVs. In this chapter, we will present basic protocols for gel-based and quantitative
MS-based approaches applied to the study of EVs.
Key words Extracellular vesicles, Platelets, 2D-DIGE, Label-free LC-MS/MS
1 Introduction
Over the past decade, the interest in extracellular vesicles (EVs) as a
novel source of biomarkers has increased dramatically due to their
implication in cell-to-cell communication, cellular activation,
inflammation, hemostasis, and disease development and progression [1]. EVs are a heterogeneous population of cell-derived circulating vesicles. However, there is some controversy on
nomenclature and sizes of the different types of vesicles. For this
reason, the International Society for Extracellular Vesicles (ISEV)
has provided some criteria to classify EVs, categorizing them into
three groups: microvesicles (MVs), exosomes, and apoptotic bodies
[2]. MVs (100–1000 nm) are formed by outward blebbing of the
plasma membrane and subsequent fission of plasma membrane
blebs. In contrast, exosomes (30–100 nm) are formed by multivesicular bodies being stored on the cytoplasm upon stimulus. The
multivesicular bodies fusion with the plasma membrane and release
their content (exosomes) to the extracellular environment
[3, 4]. Lastly, apoptotic bodies (500–4000 nm) are formed as a
result of the induction of cellular apoptosis [5].
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
13