Mass spectrometry of transferrin is an established method for the detection and diagnosis of congenital disorder of glycosylation (CDG). Transferrin is an 80 kDa glycoprotein and the glycoform at two ...N‐glycosylation sites is comprised of a di‐sialylated biantennary oligosaccharide as the major form and minor species with fucosylated or triantennary structures. Rapid CDG screening is carried out by MS of native transferrin. On the other hand, MS of glycopeptides enables site‐specific determination of glycoforms, and the affinity‐based enrichment of glycopeptides from a complex mixture of proteolytic peptides facilitates efficient analysis. MS of glycopeptides reveals the presence of immature glycoforms even in healthy individuals, indicating that the diagnosis of CDG based on molecular phenotypes requires quantitative evaluation. In this technical note, the aberrant glycosylation profiles of CDG cases are presented to shed light on the MS of native transferrin and glycopeptides from the viewpoint of clinical glycoproteomics.
Congenital disorder of glycosylation (CDG), formerly representing a group of diseases due to defects in the biosynthetic pathway of protein N-glycosylation, currently covers a wide range of disorders ...affecting glycoconjugates. Since its first application to serum transferrin from a CDG patient with phosphomannomutase-2 deficiency in 1992, mass spectrometry (MS) has been playing a key role in identification and characterization of glycosylation defects affecting glycoproteins. MS of native transferrin detects a lack of glycans characteristic to the classical CDG-I type of molecular abnormality. Electrospray ionization MS of native transferrin, especially, allows glycoforms to be analyzed precisely but requires basic knowledge regarding deconvolution of multiply-charged ions which may generate ghost signals upon transformation into a singly-charged form. MS of glycopeptides from tryptic digestion of transferrin delineates site-specific glycoforms and reveals a delicate balance of donor/acceptor substrates or the conformational effect of nascent proteins in cells. Matrix-assisted laser desorption ionization MS of apolipoprotein C-III is a simple method of elucidating the profiles of mucin-type core 1 O-glycans including site occupancy and glycoforms. In this technological review, the principle and pitfalls of MS for CDG are discussed and mass spectra of various types of CDG are presented.
Congenital disorders of glycosylation (CDG), an increasingly recognized group of diseases that affect glycosylation, comprise the largest known subgroup of approximately 100 responsible genes related ...to N-glycosylation. This subgroup presents various molecular abnormalities, of either the CDG-I or the CDG-II type, attributable to a lack of glycans or abnormal glycoform profiles, respectively. The most effective approach to identifying these N-glycosylation disorders is mass spectrometry (MS) using either released glycans, intact glycoproteins or proteolytic peptides as analytes. Among these, MS of tryptic peptides derived from transferrin can be used to reliably identify signature peptides that are characteristic of CDG-I and II. In the present study, matrix-assisted laser desorption/ionization (MALDI) MS was applied to various N-glycosylation disorders including ALG1-CDG, B4GALT1-CDG, SLC35A2-CDG, ATP6V0A2-CDG, TRAPPC11-CDG and MAN1B1-CDG. This method does not require the prior enrichment of glycopeptides or chromatographic separation, and thus serves as a practical alternative to liquid chromatography-electrospray ionization MS. The signature peptides are biomarkers of CDG.
Folic acid supplementation and folate-rich diets are recommended for women of childbearing age worldwide to prevent congenital anomalies. We aimed to determine the current status of folic acid ...supplementation among pregnant Japanese women and identify means to increase the intake of these supplements.
Cross-sectional study.
A total of 1862 pregnant women who consulted the perinatal centre from September 2014 to December 2015 completed a questionnaire concerning knowledge about folic acid, sources of information and the use of folic acid supplements.
Osaka Medical Center and Research Institute for Maternal and Child Health (Japan).
In our study population, only 20·5 % of pregnant women took folic acid supplements periconceptionally even though 70·4 % knew about the protective effect of folic acid. A multivariate analysis demonstrated that age ≥35 years (OR=2·80; 95 % CI 1·24, 6·29) and knowledge of the benefits of folic acid (OR=2·64; 95 % CI 1·92, 3·62) were associated with periconceptional folic acid use, and multiparity was negatively associated with such use. Compared with those who took folic acid supplements periconceptionally, women who did not take supplements received information through passive and less interactive media.
Although folic acid awareness was relatively high among pregnant Japanese women, folic acid supplementation before conception was insufficient. To increase the intake of folic acid supplements in countries in which foods are not fortified with folic acid, an effective public health approach promoting behavioural change is necessary for women of reproductive age.
Profiling of glycans requires both characterization of structure and determination of the relative abundance of each glycan. Label-free approaches enable facile and efficient profiling, while ...detailed structures and precise quantitation require derivatization. For glycan profiling by mass spectrometry, correlating the ion abundance in the mass spectrum to the content of each glycoform in the sample is acceptable, when one has adequate knowledge of ionization mode and ionization efficiency in mass spectrometry. Glycopeptide is a suitable analyte for this label-free approach.
Electron transfer dissociation (ETD) has been used for peptide sequencing. Since ETD preferentially produces the c′/z • fragment pair, peptide sequencing is generally performed by interpretation of ...mass differences between series of consecutive c′ and z • ions. However, the presence of free cysteine residues in a precursor promotes peptide bond cleavage, hindering interpretation of the ETD spectrum. In the present study, the divalent group XII metals, such as Zn2+, Cd2+ and Hg2+, were used as charge carriers to produce metal-peptide complexes. The thiol group is deprotonated by complexation with the group XII metal. The formation of b and y′ ions was successfully suppressed by using the zinc-peptide complex as a precursor, indicating Zn2+-aided ETD to be a useful method for sequencing of cysteine-containing peptides. By contrast, ETD of Cd2+– and Hg2+–peptide complexes mainly led to SH2 loss and radical cation formation, respectively. These processes were mediated by recombination energy between the metal cation and an electron. The presence of monovalent cadmium and neutral mercury in ETD products was confirmed by MS3 analysis with collision-induced dissociation.
Congenital disorders of glycosylation (CDG) constitute a group of diseases affecting N-linked glycosylation pathways. The classical type of CDG, now called CDG-I, results from deficiencies in the ...early glycosylation pathway for biosynthesis of lipid-linked oligosaccharide and its transfer to proteins in endoplasmic reticulum, while the CDG-II diseases are caused by defects in the subsequent processing steps. Mass spectrometry (MS) produced a milestone in CDG research, by localizing the CDG-I defect to the early glycosylation pathway in 1992. Currently, MS of transferrin, either by electrospray ionization or matrix-assisted laser desorption/ionization, plays the central role in laboratory screening of CDG-I. On the other hand, the glycopeptide analysis recently developed for site-specific glycans of glycoproteins allows detailed glycan analysis in a high throughput manner and will solve problems in CDG-II diagnosis. These techniques will facilitate studying CDG, a field now expanding to O-linked glycosylation and to acquired as well as inherited conditions that can affect protein glycosylation.
Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes ...muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD) is cytidine diphosphate ribitol (CDP-Rbo) synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases.
Display omitted
•Ribitol 5-phosphate is a functional glycan unit in mammals•α-dystroglycan function requires tandem ribitol 5-phosphate structure•Muscular dystrophy proteins are involved in ribitol 5-phosphate glycosylation•Supplementation with ribitol 5-phosphate metabolites may be a therapeutic strategy
Kanagawa et al. show that ribitol 5-phophate is a functional glycan unit in mammals and that defects in its post-translational modification pathway are associated with muscular dystrophy.
In glycoproteomics, key structural issues, protein identification, locations of glycosylation sites, and evaluation of the glycosylation site microheterogeneity should be easily evaluated in a large ...number of glycoproteins, while mass spectrometry (MS) provides substantial information about individual purified glycoproteins. Considering that structural issues are elucidated by studying glycopeptides and that the tandem MS of a tryptic peptide composed of several amino acid residues is enough for protein identification, construction of an MS-based method handling tryptic glycopeptides would be of considerable benefit in research. To this end, a simple and efficient method, utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides, was successfully applied to the isolation of tryptic glycopeptides. Both peptide and oligosaccharide structures were elucidated by multiple-stage tandem MS (MS n ) of the ions generated by matrix-assisted laser desorption/ionization (MALDI), as follows. The MALDI ion trap mass spectrum of a tryptic glycopeptide mixture from N-linked glycoproteins was composed of the M + H+ ions of component glycopeptides. Collision-induced dissociation (CID) of the glycopeptide M + H+ ion generated saccharide-spaced peaks, with an interval of, for example, 146, 162, and 203 Da, and their fragment ions corresponding to the peptide and peptide + N-acetylglucosamine (GlcNAc) species in the MS2 spectrum. The saccharide-spaced ladder served to outline oligosaccharide structures, which were then selected as precursors for subsequent MS n analyses. The peptide or peptide + GlcNAc ions in the MS2 spectrum or the corresponding ions abundant in the MS1 spectrum were subjected to CID for determination of peptide sequences, to identify proteins and their glycosylation sites. The strategy, isolation of glycopeptides followed by MS n analysis, efficiently characterized the structures of β2-glycoprotein I with four N-glycosylation sites and was applied to an analysis of total serum glycoproteins.
Congenital disorders of glycosylation (CDG) are inherited metabolic diseases that affect the synthesis of glycoconjugates. Defects in mucin-type O-glycosylation occur independently or in combination ...with N-glycosylation disorders, and the profiling of the O-glycans of apolipoprotein CIII (apoCIII) by mass spectrometry (MS) can be used to support a diagnosis. The biomarkers are site occupancy and sialylation levels, which are indicated by the content of non-glycosylated apoCIII0a isoform and by the ratio of monosialylated apoCIII1 to disialylated apoCIII2 isoforms, respectively. In this report, electrospray ionization (ESI) quadrupole MS of apoCIII was used to identify these biomarkers. Among the instrumental parameters, the declustering potential (DP) induced the fragmentation of the O-glycan moiety including the Thr–GalNAc linkage, resulting in an increase in apoCIII0a ions. This incurs the risk of creating a false positive for reduced site occupancy. The apoCIII1/apoCIII2 ratio was substantially unchanged despite some dissociation of sialic acids. Therefore, appropriate DP settings are especially important when transferrin, which requires a higher DP, for N-glycosylation disorders is analyzed simultaneously with apoCIII in a single ESI MS measurement. Finally, a reference range of diagnostic biomarkers and mass spectra of apoCIII obtained from patients with SLC35A1-, TRAPPC11-, and ATP6V0A2-CDG are presented.
PDF
