Dataset Information

Use of the proteomics approach to study endometrial cancer.

ABSTRACT: Endometrial cancer (EC) is the second most frequent gynecological malignant tumor in postmenopausal women. Pathogenic mechanisms related to the onset and development of disease are still unknown. In this study, we aimed to characterize the EV proteome by combining Data-Independent Acquisition (DIA) acquisition, in albumin-depleted serum EVs to identify dysregulated proteins and enzymes associated with the disease. A deep proteomics analysis with advanced computational tools allowed us to identify a large number of proteins in serum albumin-depleted extracellular vesicles (EVs) from 10 patients with EC compared to 10 healthy controls. This is the largest number of proteins identified in EC serum EVs. After quantification and statistical analysis, we identified 373 significantly (p < 0.05) dysregulated proteins involved in neutrophil and platelet degranulation pathways. A more detailed bioinformatics analysis revealed 61 dysregulated enzymes related to metabolic and catabolic pathways linked to tumor invasion. Our bioinformatic analysis identified 49 metabolic and catabolic pathways related to tumor growth. MTD project_tag Cancer (B/D-HPP)

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

DISEASE(S): Endometrial Cancer

SUBMITTER: Michelangelo Aloisio

LAB HEAD: Blendi Ura

PROVIDER: PXD058193 | Pride | 2025-05-07

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	DIA_dia_elaborated_serum_exosome.xlsx	Xlsx
	checksum.txt	Txt
	wetransfer_ctrl1-2_2024-11-22_0840.zip	Other
	wetransfer_ctrl3-4_2024-11-22_0914.zip	Other
	wetransfer_ctrl5-6_2024-11-22_1023.zip	Other

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Publications

A Pilot Study of Exosome Proteomic Profiling Reveals Dysregulated Metabolic Pathways in Endometrial Cancer.

Kharrat Feras F Capaci Valeria V Conti Andrea A Golino Valentina V Campiglia Pietro P Balasan Nour N Aloisio Michelangelo M Licastro Danilo D Monasta Lorenzo L Caponneto Federica F Beltrami Antonio Paolo AP Romano Federico F di Lorenzo Giovanni G Ricci Giuseppe G Ura Blendi B

Biomedicines 20250103 1

<b>Background/Objectives</b>: Endometrial cancer (EC) is the second most frequent gynecological malignant tumor in postmenopausal women. Pathogenic mechanisms related to the onset and development of the disease are still unknown. To identify dysregulated proteins associated with EC we exploited a combined in vitro/in silico approach analyzing the proteome of exosomes with advanced MS techniques and annotating their results by using Chymeris1 AI tools. <b>Methods</b>: To this aim in this pilot st ...[more]

PMID: 39857679

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Project description:Background Plasma proteins can be modified by addition of sugar residues by non-enzymatic glycation as well as glycosylation. While glycation is a hallmark of hyperglycemia, glycosylation is a complex, enzyme-catalyzed post-translational modification by heterogeneous sugar chains and is present on a majority of plasma proteins. N-linked glycosylation occurs on asparagine residues occurring predominantly on a canonical N-glycosylation motif (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported to be glycosylated less frequently. Albumin is the most abundant protein in human plasma whose glycation has been implicated in end-organ damage in advanced diabetes mellitus, and as such, has both diagnostic and therapeutic implications. However, albumin has long been regarded as a non-glycosylated protein owing to the absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated with two possible attached glycans. Methods To investigate N-linked glycosylation of abundant serum proteins in greater detail, we passed healthy donor samples through a multiple affinity removal spin (MARS14) column followed by trypsin digestion of bound proteins and subsequent glycopeptide enrichment by size-exclusion chromatography (SEC) or mixed-mode anion-exchange (MAX), in parallel. Global analysis of intact glycopeptides was performed by LC-MS/MS to evaluate potential glycosylation at canonical as well as non-canonical sites. PNGase F treatment was carried out to confirm N-linked glycosylation at non-canonical sites Asn-X-Cys/Val. Glycopeptides on albumin at non-canonical sites were further characterized by MS3 fragmentation from immunoprecipitated albumin to confirm the attached glycans. To assess if the observed glycosylation was a general phenomenon, targeted analysis using parallel reaction monitoring (PRM) was carried out on an independent set of twenty human serum samples. Finally, to test whether albumin glycosylation is unique to human albumin or is a general phenomenon, bovine and rabbit serum albumins were subjected to MAX chromatography and LC-MS/MS analysis. Results We report that human albumin is modified by N-linked glycosylation at two sites, Asn68(-Glu-Val) and Asn123-(Glu-Cys), both of which occur in non-canonical N-glycosylation motifs. We detected N-glycopeptides at both sites bearing four complex mono- and bi-antennary N-linked glycans, with Hex5HexNAc4NeuAc2 and Hex5HexNAc4NeuAc1 being the most abundant glycans. These findings were validated by analyzing deglycosylated peptides and MS3-based fragmentation of glycopeptides. Glycosylation at both sites was confirmed by targeted MS in twenty donor samples. Finally, we report that the Asn residue on bovine and rabbit serum albumins that corresponds to the highly conserved Asn123 in human albumin is also N-glycosylated with complex sialylated glycans similar to those observed in human albumin. Conclusions Albumin is a conserved glycoprotein with multiple N-linked glycosylation sites. This glycosylation of albumin has potential functional implications and applications in medicine.