258
Current MS approaches for the UE glycoproteome investigation are limited by
the low amount of UE proteins; furthermore, the presence of Uromodulin (UMOD),
the most abundant glycoprotein in urine, hampers the UE recovery and the study of
the other glycoproteins. These evidences arise a double technical challenge: primarily, the reduction of UMOD contamination, and secondly the search of a suitable
strategy to investigate the UE glycoproteome. Thus, we tested different experimental procedures, starting from normal urine specimens, in order to improve the sample preparation and achieve a better MS analysis.
First, we performed the UE isolation through multistep differential centrifugations, trying to reduce UMOD presence by exploiting some chemical-physical features of this protein (oxidative state, solubility, size). The best compromise between
the UE yield and the UMOD depletion emerged to be the addition of ZnSO4 to
frozen urine specimens prior to UE purification. Then, we set up an optimised
N-Glyco-FASP (Filter Aided Sample Preparation), a recently developed MS
approach based on lectin-enrichment able to capture UE glycoproteins [3]. We compared the efficiency of different types of filters (Microcon vs Amicon) in order to
improve the UE peptides and glycopeptides recovery.
Despite the small amount of starting sample (urine volume <40 ml; UE proteins
<100 μg), we confirmed the correct glycopeptides enrichment, highlighting that our
combined approach is suitable for the study of the UE glycoproteome. It would be
interesting to glyco-characterise UE from patients affected by different type of kidney pathologies (Renal Cell Carcinoma and Idiopathic Nephrotic Syndrome), to
bring out the remarkable impact of this PTM within the pathological mechanism of
these diseases.
References
1. Taniguchi N, Kizuka Y (2015) Glycans and cancer: role of N-glycans in cancer biomarker,
progression and metastasis, and therapeutics. Adv Cancer Res 126:11–51
2. Williams C, Royo F, Aizpurua-Olaizola O, Pazos R, Boons G, Reichardt N, Falcon-Perez JM
(2018) Glycosylation of extracellular vesicles: current knowledge, tools and clinical perspectives. J Extracell Vesic 7(1):1442985
3. Deeb S, Cox J, Schmidt-Supprian M, Mann M (2014) N-linked glycosylation enrichment
for in-depth cell surface proteomics of diffuse large B-cell lymphoma subtypes. Mol Cell
Proteomics 13(1):240–251
L. Santorelli et al.
Current MS approaches for the UE glycoproteome investigation are limited by
the low amount of UE proteins; furthermore, the presence of Uromodulin (UMOD),
the most abundant glycoprotein in urine, hampers the UE recovery and the study of
the other glycoproteins. These evidences arise a double technical challenge: primarily, the reduction of UMOD contamination, and secondly the search of a suitable
strategy to investigate the UE glycoproteome. Thus, we tested different experimental procedures, starting from normal urine specimens, in order to improve the sample preparation and achieve a better MS analysis.
First, we performed the UE isolation through multistep differential centrifugations, trying to reduce UMOD presence by exploiting some chemical-physical features of this protein (oxidative state, solubility, size). The best compromise between
the UE yield and the UMOD depletion emerged to be the addition of ZnSO4 to
frozen urine specimens prior to UE purification. Then, we set up an optimised
N-Glyco-FASP (Filter Aided Sample Preparation), a recently developed MS
approach based on lectin-enrichment able to capture UE glycoproteins [3]. We compared the efficiency of different types of filters (Microcon vs Amicon) in order to
improve the UE peptides and glycopeptides recovery.
Despite the small amount of starting sample (urine volume <40 ml; UE proteins
<100 μg), we confirmed the correct glycopeptides enrichment, highlighting that our
combined approach is suitable for the study of the UE glycoproteome. It would be
interesting to glyco-characterise UE from patients affected by different type of kidney pathologies (Renal Cell Carcinoma and Idiopathic Nephrotic Syndrome), to
bring out the remarkable impact of this PTM within the pathological mechanism of
these diseases.
References
1. Taniguchi N, Kizuka Y (2015) Glycans and cancer: role of N-glycans in cancer biomarker,
progression and metastasis, and therapeutics. Adv Cancer Res 126:11–51
2. Williams C, Royo F, Aizpurua-Olaizola O, Pazos R, Boons G, Reichardt N, Falcon-Perez JM
(2018) Glycosylation of extracellular vesicles: current knowledge, tools and clinical perspectives. J Extracell Vesic 7(1):1442985
3. Deeb S, Cox J, Schmidt-Supprian M, Mann M (2014) N-linked glycosylation enrichment
for in-depth cell surface proteomics of diffuse large B-cell lymphoma subtypes. Mol Cell
Proteomics 13(1):240–251
L. Santorelli et al.