So far, extensive research has been carried out to characterize
the content of EVs. EVs have a large variety of proteins, sugars,
lipids, and genetic material (mRNA, microRNA, rRNA, and tRNA)
[6]. The recent development of more robust and sensitive proteomic strategies has allowed a better knowledge of EVs cargo. Indeed,
it has been shown that the protein profile of circulating EVs
changes when transitioning from health to disease. For this reason,
the role of EVs has gained significant interest in the clinical practice
as promising targets/sources of biomarkers for the diagnosis, prognosis, and treatment of several pathologies [7].
In recent years, our group has provided an overall picture of the
EV proteome in several cardiovascular and cardiometabolic diseases. Initially, we identified novel potential EV-related biomarkers
for ST-elevation myocardial infarction (STEMI) by comparing the
proteome of EVs from STEMI patients to matched stable coronary
artery disease (CAD) controls [8]. More recently, our group carried
out a study comparing the proteome of circulating EVs from morbidly obese patients and lean individuals following two different
proteomic approaches: 2D-DIGE-based and label-free MS-based
proteomics. In that way, we identified a panel of biomarkers related
to an increased cardiovascular risk in obesity [9]. The above
approaches will be the basis for the protocols here presented.
In this chapter, we will describe basic protocols of EV isolation
and subsequent proteomic analysis. Moreover, we will focus on
gel-based and quantitative MS-based approaches applied to the
study of EVs in an attempt to identify novel EV biomarkers in a
clinical context.
2 Materials
2.1 Blood Collection
1. 3.2% sodium citrate tubes.
2. ACD: 78 mM citric acid, 96.6 mM trisodium citrate, and
111 mM glucose.
3. Eppendorf 5702 centrifuge and Eppendorf 5415 R.
2.2 EV Isolation
1. Beckman XL-100K Ultracentrifuge.
2. SW55 Ti rotor.
3. Ultra-clear 344057 tubes.
4. HEPES: 10 mM HEPES, 5 mM KCl, 1 mM MgCl 2 , 136 mM
NaCl (pH 7.4).
5. 0.25 M KBr.
6. PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 ·12H 2 O,
2 mM KH 2 PO 4 , 1 L H 2 Omq (pH 7.4).
14
Maria N. Barrachina and A ´ ngel Garcı ´a
the content of EVs. EVs have a large variety of proteins, sugars,
lipids, and genetic material (mRNA, microRNA, rRNA, and tRNA)
[6]. The recent development of more robust and sensitive proteomic strategies has allowed a better knowledge of EVs cargo. Indeed,
it has been shown that the protein profile of circulating EVs
changes when transitioning from health to disease. For this reason,
the role of EVs has gained significant interest in the clinical practice
as promising targets/sources of biomarkers for the diagnosis, prognosis, and treatment of several pathologies [7].
In recent years, our group has provided an overall picture of the
EV proteome in several cardiovascular and cardiometabolic diseases. Initially, we identified novel potential EV-related biomarkers
for ST-elevation myocardial infarction (STEMI) by comparing the
proteome of EVs from STEMI patients to matched stable coronary
artery disease (CAD) controls [8]. More recently, our group carried
out a study comparing the proteome of circulating EVs from morbidly obese patients and lean individuals following two different
proteomic approaches: 2D-DIGE-based and label-free MS-based
proteomics. In that way, we identified a panel of biomarkers related
to an increased cardiovascular risk in obesity [9]. The above
approaches will be the basis for the protocols here presented.
In this chapter, we will describe basic protocols of EV isolation
and subsequent proteomic analysis. Moreover, we will focus on
gel-based and quantitative MS-based approaches applied to the
study of EVs in an attempt to identify novel EV biomarkers in a
clinical context.
2 Materials
2.1 Blood Collection
1. 3.2% sodium citrate tubes.
2. ACD: 78 mM citric acid, 96.6 mM trisodium citrate, and
111 mM glucose.
3. Eppendorf 5702 centrifuge and Eppendorf 5415 R.
2.2 EV Isolation
1. Beckman XL-100K Ultracentrifuge.
2. SW55 Ti rotor.
3. Ultra-clear 344057 tubes.
4. HEPES: 10 mM HEPES, 5 mM KCl, 1 mM MgCl 2 , 136 mM
NaCl (pH 7.4).
5. 0.25 M KBr.
6. PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 ·12H 2 O,
2 mM KH 2 PO 4 , 1 L H 2 Omq (pH 7.4).
14
Maria N. Barrachina and A ´ ngel Garcı ´a