A complement C4-derived glycopeptide is a biomarker for PMM2-CDG

Abstract

BACKGROUNDDiagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG.METHODSUsing multistep mass spectrometry-based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag-based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion.RESULTSOf the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and 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, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2.CONCLUSIONComplement C4-derived Man5GlcNAc2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs.FUNDINGU54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance).

Keywords: Genetic diseases; Genetics; Glycobiology; Metabolism; Proteomics.

Figure 1. Glycopeptide alterations in PMM2-CDG.

(A) Overview of protein N-glycosylation and role of phosphomannomutase (encoded by PMM2). (B) Experimental schematic for TMT labeling–based quantitative glycoproteomics and proteomics. TMT, tandem mass tag. (C) Principal component analysis (PCA) plot for TMT-based glycoproteomics data; samples from individuals affected with PMM2-CDG (n = 7) and controls (n = 7) are represented by circles as indicated. (D) Volcano plot showing global glycosylation changes in individuals with PMM2-CDG (n = 7) in comparison with controls (n = 7); each point represents a glycopeptide with the glycoprotein from which it is derived labeled in black and the amino acid site of glycosylation in red; where the detected glycopeptide backbones contain multiple known glycosylation sites, the nonglycosylated site is labeled in blue; putative structures are shown using symbol nomenclature for glycans (SNFG) and represent glycan composition inferred from mass spectrometry data (63).

Figure 2. Site-specific alterations in glycopeptide abundance from TMT-based experiments in the discovery set.

(A) Box plots representing levels of significantly altered glycopeptides; the glycoprotein from which the glycopeptide is derived is labeled in black and the amino acid site of glycosylation in red; putative structures are shown using SNFG and represent glycan composition inferred from mass spectrometry data (63). The box plots depict minimum and maximum values (whiskers), upper and lower quartiles, and median. The length of the box represents the interquartile range. *=q < 0.05, **=q < 0.01, ***=q < 0.001. (B) Heatmap showing the most significantly increased (q < 0.05) glycopeptides in PMM2-CDG. (C) Heatmap showing the most significantly decreased (q < 0.05) glycopeptides in PMM2-CDG; glycopeptides are represented by protein followed by the amino acid site of glycosylation and glycan composition. The q values in A–C were calculated by t test with multiple testing using Benjamini-Hochberg procedure. PMM2-CDG (n = 7), controls (n = 7). Hex, hexose; HexNAc, N-acetylglucosamine; NeuAc, N-acetylneuraminic acid; Fuc, fucose.

Figure 3. Microheterogeneity in glycosylation for selected proteins from TMT-based experiments in the discovery set.

Box plots showing relative abundance of glycopeptides bearing different glycans at the same site in selected proteins (A) complement C4 Asn²²⁶ and (B) ceruloplasmin Asn¹³⁸. (C) Representative haptoglobin-derived glycopeptides containing Asn²⁰⁷ and Asn²¹¹ but with glycan occupancy only at Asn²⁰⁷, with Asn²¹¹ unoccupied. (D) Representative haptoglobin-derived glycopeptides containing Asn²⁰⁷ and Asn²¹¹ but with glycan only at Asn²¹¹, with Asn²⁰⁷ unoccupied. (E) Representative haptoglobin-derived glycopeptides containing Asn²⁰⁷ and Asn²¹¹ with glycosylation at both Asn²⁰⁷ and Asn²¹¹; putative structures are shown using SNFG and represent total composition of glycan(s) inferred from mass spectrometry data (63). The box plots depict minimum and maximum values (whiskers), upper and lower quartiles, and median. The length of the box represents the interquartile range. PMM2-CDG (n = 7), controls (n = 7); *=q < 0.05, **=q < 0.01, ***=q < 0.001; the q values in A–E were calculated by t test with multiple testing using Benjamini-Hochberg procedure.

Figure 4. Profiling N-glycosylation using MAX cartridge–based enrichment and single-shot MS analysis.

(A) Strategy for MAX-based enrichment and analysis. (B) Heatmap showing glycopeptides identified by label-free analysis using discovery methods; glycopeptides are represented by protein followed by the amino acid site of glycosylation and glycan composition. PMM2-CDG (n = 17), controls (n = 17). Hex, hexose; HexNAc, N-acetylglucosamine; NeuAc, N-acetylneuraminic acid. Fuc, fucose. (C) Annotated MS/MS spectrum for the Man₅GlcNAc₂ glycopeptide from complement C4 at site Asn²²⁶; putative structures are shown using SNFG and represent glycan composition inferred from MS data (63). (D) Dot plot representing normalized peak intensity values for the Man₅GlcNAc₂ glycopeptide from complement C4 at site Asn²²⁶ in PMM2-CDG and control samples. Each dot represents abundance in a sample from an individual, and the horizontal bar represents the median; special cases are labeled and shown separately as follows: “Before treatment”: sample 3 (n = 1) was donated by an individual affected with PMM2-CDG before treatment with epalrestat; “After epalrestat treatment”: sample 15 (n = 1) was donated by the same individual at 6 months of therapy with epalrestat; “After liver transplantation”: sample 24 (n = 1) was donated by an affected individual after liver transplantation; other PMM2-CDG (n = 15); controls (n = 17). ND, not detected.

Figure 5. Detection of Man₅GlcNAc₂ glycopeptide from complement C4 by targeted PRM assay.

(A) Strategy for MAX-based enrichment and targeted analysis of glycopeptides. (B) Representative extracted ion chromatogram showing abundance of Man₅GlcNAc₂ glycopeptide from complement C4 at Asn²²⁶ in an individual with PMM2-CDG. (C) Representative extracted ion chromatogram at the same retention time in a control sample. (D) Dot plot showing abundance of the Man₅GlcNAc₂ glycopeptide from complement C4 at Asn²²⁶ in PMM2-CDG (n = 8) and control (n = 8) samples used for development of PRM assay. The dots represent abundance in each sample, and the horizontal bars represent, from top to bottom, the maximum value, median, and minimum value, respectively.

Figure 6. Detection of Man₅GlcNAc₂ glycopeptide from complement C4 at Asn²²⁶ by blinded analysis correctly identifies individuals with PMM2-CDG.

Representative Skyline plots showing glycopeptide fragment peaks from selected samples in the blinded set. Targeted analysis was performed on 16 samples by PRM by operators blinded to the identity of the samples. (A–D) Samples identified to be from individuals with PMM2-CDG. (E–H) Samples identified to be from controls.