Dataset Information

N-glycoproteomics and proteomics in NGLY1 deficient patient-derived dermal fibroblasts

ABSTRACT: In the biological systems, several genes are involved in protein glycosylation and deglycosylation pathways. Congenital disorders of deglycosylation (CDDG) are a set of disorders which occur due to the defect in genes involved in deglycosylation pathways. The only known CDDG so far is the defect in N-glycanase 1 (NGLY1), which primary function is to cleave the N-glycans from misfolded proteins prior to their proteasomal degradation. We used TMT-based N-glycoproteomics and proteomics on patient derived NGLY1 deficient and control fibroblasts to characterize the alteration in glycoproteome and proteome. 24 fractions of enriched glycopeptides after size exclusion chromatography (SEC) and 24 fractions after basic reverse phase liquid chromatography (bRPLC) were analyzed by LC-MS/MS for glycoproteomics and proteomics, respectively. We identified a total of 3,255 N-glycopeptides which were quantified on 550 glycosylation sites of 407 glycoproteins. A site specific aberrant glycosylation was observed for several extracellular matrix and cell adhesion proteins. By using quantitative proteomics, we detected 8,041 proteins. The alteration in expression of several proteins separated the affected individuals and controls. This is the first glycoproteomic study in patient derived NGLY1-CDDG fibroblasts. The glycoproteomics and proteomics analysis in NGLY1-CDDG provides the potential biomarkers and will increase our general understanding of its pathogenesis.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Fibroblast

DISEASE(S): Congenital Disorder Of Glycosylation

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD034364 | Pride | 2023-05-10

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	040621_Rohit_NGLY1_NGP_TMT_SEC_B01.raw	Raw
	040621_Rohit_NGLY1_NGP_TMT_SEC_B02.raw	Raw
	040621_Rohit_NGLY1_NGP_TMT_SEC_B03.raw	Raw
	040621_Rohit_NGLY1_NGP_TMT_SEC_B04.raw	Raw
	040621_Rohit_NGLY1_NGP_TMT_SEC_B05.raw	Raw

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Publications

N-glycoproteomics reveals distinct glycosylation alterations in NGLY1-deficient patient-derived dermal fibroblasts.

Budhraja Rohit R Saraswat Mayank M De Graef Diederik D Ranatunga Wasantha W Ramarajan Madan G MG Mousa Jehan J Kozicz Tamas T Pandey Akhilesh A Morava Eva E

Journal of inherited metabolic disease 20221004 1

Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retro-translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylatio ...[more]

PMID: 36102038

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Project description:The most common congenital disorder of glycosylation (CDG) is caused by pathogenic variants in PMM2 (PMM2-CDG) that reduce phosphomannomutase activity and impair N-glycan synthesis. Patients present early in life with multi-system disabilities. Traditional diagnosis relies on measuring carbohydrate-deficient transferrin (CDT) and confirmation by genetic testing. However, tests for CDT can be normal in some patients, or normalize with age. Because patients also show subtle alterations in serum glycan structures, we considered that site-specific glycosylation changes might be diagnostic. We analyzed serum from 35 individuals with PMM2-CDG for discovery and validation of glycoproteomic changes. We carried out mass spectrometry-based discovery studies to profile the glycoproteome in an initial set of well-characterized affected individuals and matched controls. Next, we developed a streamlined method for analyzing additional affected individuals. Finally, targeted mass spectrometry was used to validate selected N-glycopeptides in an independent set of samples in a blinded fashion. Of the 3,342 N-glycopeptides derived from 284 glycoproteins identified in discovery experiments, individuals with PMM2-CDG exhibited a general decrease in complex-type N-glycans and an increase in truncated, mannose-rich and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man5GlcNAc2, which was not detected in controls. Notably, this glycopeptide was also detected in five affected individuals with normal CDT, including one individual after liver transplant, two individuals with a known genetic variant associated with mild disease and two individuals with well characterized variants, indicating that this glycopeptide is more sensitive than CDT. Finally, this marker was also detected by targeted analysis in two individuals with variants of uncertain significance (VUS) in PMM2 gene. Overall, we believe that C4-derived Man5GlcNAc2 glycopeptide could serve as a novel next-generation biomarker for more accurate diagnosis and tracking progress of patients in therapeutic trials.