257
© Springer Nature B.V. 2020
G. Sindona et al. (eds.), Toxic Chemical and Biological Agents, NATO Science
for Peace and Security Series A: Chemistry and Biology,
https://doi.org/10.1007/978-94-024-2041-8_26
Chapter 26
Investigation of the N-Glycoproteome
in the Urinary Exosomes: Technical
Challenges
Lucia Santorelli, Elisa Barigazzi, M. Pitto, and F. Raimondo
Abstract Urinary Exosomes (UE) are membranous nanometer-sized vesicles that
can originate from endothelial cells, podocytes or tubular epithelial cells. Their
molecular composition depends upon the type, and even status, of the producer cell.
Their presence in urine makes them readily accessible, representing a liquid biopsy,
non-invasive modality capable to provide diagnostic and prognostic information
about kidney health state. Additionally, the UE proteome includes many species that
are N-glycomodified. N-glycosylation is one of the most complex and frequent protein post-translational modifications, playing diverse biological roles. Additionally, it
is reported that disease-specific glycosylation profiles of circulating microvesicles
were detected in autosomal dominant polycystic kidney disease and ovarian carcinoma. This study is set in this context. Our aim is to provide the best MS-sample
preparation protocol to study the UE glycosylated protein content, focusing our
efforts on the enrichment of the glycosylated species and simultaneously depleting
the most abundant contaminant protein (Uromodulin).
Protein N-glycosylation is a biologically important post-translational modification
(PTM), often altered on both glycosites and glycans. Its aberrant changes are closely
associated with several diseases, including renal dysfunction and urologic malignancies [1].
In this context, Urinary Exosomes (UE), nanovesicles representing a molecular
snapshot of the parental cell, are enriched in glycosylated renal proteins; in particular, their surface presents a complex glycoprotein pattern that can be considered a
specific glycan signature [2]. While it is widely known the connection between UE
and the pathophysiologic status of the origin cell, the characterisation of their glycoprotein content requires the development of new analytical procedures.
L. Santorelli (*) · E. Barigazzi · M. Pitto · F. Raimondo
School of Medicine and Surgery, University of Milano – Bicocca, Milan, Italy
e-mail: l.santorelli@campus.unimib.it; e.barigazzi@campus.unimib.it
© Springer Nature B.V. 2020
G. Sindona et al. (eds.), Toxic Chemical and Biological Agents, NATO Science
for Peace and Security Series A: Chemistry and Biology,
https://doi.org/10.1007/978-94-024-2041-8_26
Chapter 26
Investigation of the N-Glycoproteome
in the Urinary Exosomes: Technical
Challenges
Lucia Santorelli, Elisa Barigazzi, M. Pitto, and F. Raimondo
Abstract Urinary Exosomes (UE) are membranous nanometer-sized vesicles that
can originate from endothelial cells, podocytes or tubular epithelial cells. Their
molecular composition depends upon the type, and even status, of the producer cell.
Their presence in urine makes them readily accessible, representing a liquid biopsy,
non-invasive modality capable to provide diagnostic and prognostic information
about kidney health state. Additionally, the UE proteome includes many species that
are N-glycomodified. N-glycosylation is one of the most complex and frequent protein post-translational modifications, playing diverse biological roles. Additionally, it
is reported that disease-specific glycosylation profiles of circulating microvesicles
were detected in autosomal dominant polycystic kidney disease and ovarian carcinoma. This study is set in this context. Our aim is to provide the best MS-sample
preparation protocol to study the UE glycosylated protein content, focusing our
efforts on the enrichment of the glycosylated species and simultaneously depleting
the most abundant contaminant protein (Uromodulin).
Protein N-glycosylation is a biologically important post-translational modification
(PTM), often altered on both glycosites and glycans. Its aberrant changes are closely
associated with several diseases, including renal dysfunction and urologic malignancies [1].
In this context, Urinary Exosomes (UE), nanovesicles representing a molecular
snapshot of the parental cell, are enriched in glycosylated renal proteins; in particular, their surface presents a complex glycoprotein pattern that can be considered a
specific glycan signature [2]. While it is widely known the connection between UE
and the pathophysiologic status of the origin cell, the characterisation of their glycoprotein content requires the development of new analytical procedures.
L. Santorelli (*) · E. Barigazzi · M. Pitto · F. Raimondo
School of Medicine and Surgery, University of Milano – Bicocca, Milan, Italy
e-mail: l.santorelli@campus.unimib.it; e.barigazzi@campus.unimib.it