Multiplexed glycoproteomic analysis of glycosylation disorders by sequential yolk immunoglobulins immunoseparation and MALDI-TOF MS

This study applied yolk immunoglobulins immunoaffinity separation and MALDI-TOF MS for clinical proteomics of congenital disorders of glycosylation (CDG) and secondary glycosylation disorders [galactosemia and hereditary fructose intolerance (HFI)]. Serum transferrin (Tf) and alpha1-antitrypsin (AAT) that are markers for CDG, were purified sequentially to obtain high-quality MALDI mass spectra to differentiate single glycoforms of the native intact glycoproteins. The procedure was found feasible for the investigation of protein macroheterogeneity due to glycosylation site underoccupancy then ensuing the characterization of patients with CDG group I (N-glycan assembly disorders). Following PNGase F digestion of the purified glycoprotein, the characterization of protein microheterogeneity by N-glycan MS analysis was performed in a patient with CDG group II (processing disorders). CDG-Ia patients showed a typical profile of underglycosylation where the fully glycosylated glycoforms are always the most abundant present in plasma with lesser amounts of partially and unglycosylated glycoforms in this order. Galactosemia and HFI are potentially fatal diseases, which benefit from early diagnosis and prompt therapeutic intervention. In symptomatic patients with galactosemia and in those with HFI, MALDI MS of Tf and AAT depicts a hypoglycosylation profile with a significant increase of underglycosylated glycoforms that reverses by dietary treatment, representing a clue for diagnosis and treatment monitoring.     

Mass spectrometry of transferrin and apolipoprotein C-III for diagnosis and screening of congenital disorder of glycosylation

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.     

The underglycosylation of plasma alpha 1-antitrypsin in congenital disorders of glycosylation type I is not random

Conditions under which the glycosylation capacity of cells is limited provide an opportunity for studying the efficiency of site-specific glycosylation and the role of glycosylation in the maturation of glycoproteins. Congenital disorders of glycosylation type 1 (CDG-I) provide such a system. CDG-I is characterized by underglycosylation of glycoproteins due to defects in the assembly or transfer of the common dolichol-pyrophosphate-linked oligosaccharide precursor of asparagine-linked glycans. Human plasma alpha1-antitrypsin is normally fully glycosylated at three asparagine residues (46, 83, and 247), but un-, mono-, di-, and fully glycosylated forms of alpha1-antitrypsin were detected by 2D PAGE in the plasma from patients with CDG-I. The state of glycosylation of the three asparagine residues was analyzed in all the underglycosylated forms of alpha1-antitrypsin by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was found that asparagine 46 was always glycosylated and that asparagine 83 was never glycosylated in the underglycosylated glycoforms of alpha1-antitrypsin. This showed that the asparagine residues are preferentially glycosylated in the order 46>247>83 in the mature underglycosylated forms of alpha1-antitrypsin found in plasma. It is concluded that the nonoccupancy of glycosylation sites is not random under conditions of decreased glycosylation capacity and that the efficiency of glycosylation site occupancy depends on structural features at each site. The implications of this observation for the intracellular transport and sorting of glycoproteins are discussed.     

Laboratory diagnosis of congenital disorders of glycosylation type I by analysis of transferrin glycoforms

Congenital disorders of glycosylation (CDG) are being recognized as a rapidly growing and complex group of disorders. The pathophysiology results from depressed synthesis or remodeling of oligosaccharide moieties of glycoproteins. The ultimate result is the formation of abnormal glycoproteins affecting their structure and metabolic functions. The most thoroughly studied subset of CDG are the type I defects affecting N-glycosylation. Causal mutations occur in at least 12 different genes which encode primarily monosaccharide transferases necessary for N-glycosylation in the endoplasmic reticulum. The broad clinical presentation of these glycosylation defects challenge clinicians to test for these defects in a variety of clinical settings. The first described CDG was a phosphomannomutase deficiency (CDG-Ia). The original method used to define the glycosylation defect was isoelectric focusing (IEF) of transferrin. More recently, the use of other charge separation methods and electrospray-mass spectrometry (ESI-MS) has proven valuable in detecting type I CDG defects. By mass resolution, the under-glycosylation of transferrin is characterized as the total absence of one or both N-linked oligosaccharide. Beyond providing a new understanding of the structure of transferrin in type I CDG patients, it is adaptable to high throughput serum analysis. The use of transferrin under-glycosylation to detect the type I CDG provides limited insight into the specific site of the defect in oligosaccharide assembly since its value is constrained to observation of the final product of glycoprotein synthesis. New analytical targets and tools are converging with the clinical need for diagnosis of CDG. Defining the biosynthetic sites responsible for specific CDG phenotypes is in progress, and ten more type I defects have been putatively identified. This review discusses current methods, such as IEF and targeted proteomics using mass spectrometry, that are used routinely to test for type I CDG disorders, along with some newer approaches to define the defective synthetic sites responsible for the type I CDG defects. All diagnostic endeavors are followed by the quest for a reliable treatment. The isolated success of CDG-Ib treatment will be described with the hope that this may expand to other type I CDG disorders.     

Defect in N-glycosylation of proteins is tissue-dependent in congenital disorders of glycosylation Ia

The biochemical hallmark of Congenital Disorders of Glycosylation (CDG) including type Ia is a defective N-glycosylation of serum glycoproteins. Hypoglycosylated forms of alpha1-antitrypsin have been detected by Western blot in serum from CDG Ia patients. In contrast we were not able to detect hypoglycosylation in alpha1-antitrypsin synthesized by fibroblasts, keratinocytes, enterocytes, and leukocytes. Similarly no hypoglycosylation was detectable in a membrane-associated N-linked glycoprotein, the facilitative glucose transporter GLUT-1 and also in serum immunoglobulin G isolated from sera of CDG Ia patients. We conclude that the phenotypic expression of CDG Ia is tissue-dependent.     

Effect of glycosylation on the protein pattern in 2-D-gel electrophoresis

Single proteins, when analyzed with 2-D-PAGE, often show multiple spots due to PTMs. In gels of human body fluids, the spot patterns facilitate the assignment and identification of the proteins. We analyzed serums from patients with congenital disorders of glycosylation (CDG) in which glycoproteins are strongly impacted and exhibit highly distinguishable spot patterns compared to healthy controls. We detected a typical protein pattern for alpha1-acid glycoprotein (AGP) and transferrin (Trf) that are markers for CDG. AGP contains five glycosylation sites which results in a complex microheterogeneity of the glycoprotein. On the other hand, in Trf, a glycoprotein with only two glycosylation sites, mainly biantennary complex-type-N-linked glycans are bound. We used 2-D-PAGE, MALDI-TOF-MS, and ESI-MS for the analysis of these glycoproteins and their corresponding glycans. In AGP, the heterogenic glycosylation of the different glycosylation sites is responsible for the complex spot pattern. In contrast to AGP, the protein spots of Trf cannot be explained by glycosylation. We found strong evidence that oxidation of cysteine is responsible for the spot pattern. This study contradicts the commonly accepted assumption that the multiple protein spots of Trf observed in 2-D-PAGE are due, as in AGP, to the glycosylation of the protein.     

N-glycosylation at Asn(491) in the Asn-Xaa-Cys motif of human transferrin

Glycopeptides derived from human transferrin were exhaustively analyzed by matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry (MS). Both MS techniques clearly revealed the sequences of and the attachment sites of bi-antennary complex-type oligosaccharides, at both Asn432 and Asn630, both of which are located in a well-known motif for N-glycosylation, Asn-Xaa-Ser/Thr, but also at Asn491 in the Asn-Xaa-Cys motif. The latter has been reported to be a minor N-glycosylation site in several glycoproteins. The relative abundance of this abnormal glycosylation was estimated to be approximately 2 mol% of the transferrin preparation used in this study.     

Hypoglycosylation with increased fucosylation and branching of serum transferrin N-glycans in untreated galactosemia

Untreated classic galactosemia (galactose-1-phosphate uridyltransferase [GALT] deficiency) is known as a secondary congenital disorders of glycosylation (CDG) characterized by galactose deficiency of glycoproteins and glycolipids (processing defect or CDG-II). The mechanism of this undergalactosylation has not been established. Here we show that in untreated galactosemia, there is also a partial deficiency of whole glycans of serum transferrin associated with increased fucosylation and branching as seen in genetic glycosylation assembly defects (CDG-I). Thus galactosemia seems to be a secondary "dual" CDG causing a processing as well as an assembly N-glycosylation defect. We also demonstrated that in galactosemia patients, transferrin N-glycan biosynthesis is restored upon dietary treatment.     

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity

Protein glycosylation is a critical post-translational modification that regulates the structure, stability, and function of many proteins. Mass spectrometry is currently the preferred method for qualitative and quantitative characterization of glycosylation. However, the inherent heterogeneity of glycosylation makes its analysis difficult. Quantification of glycosylation occupancy, or macroheterogeneity, has proven to be especially challenging. Here, we used a variation of high-resolution multiple reaction monitoring (MRM^HR) or pseudo-MRM for targeted data-independent acquisition that we term SWAT (sequential window acquisition of targeted fragment ions). We compared the analytical performance of SWATH (sequential window acquisition of all theoretical fragment ions), SWAT, and SRM (selected reaction monitoring) using a suite of synthetic peptides spiked at various concentrations into a complex yeast tryptic digest sample. SWAT provided superior analytical performance to SWATH in a targeted approach. We then used SWAT to measure site-specific N-glycosylation occupancy in cell wall glycoproteins from yeast with defects in the glycosylation biosynthetic machinery. SWAT provided robust measurement of occupancy at more N-glycosylation sites and with higher precision than SWATH, allowing identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of oligosaccharyltransferase.     

A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.     

Diagnosis of congenital disorders of glycosylation type-I using protein chip technology

A method for the diagnosis of the congenital disorders of glycosylation type I (CDG-I) by SELDI-TOF-MS of serum transferrin immunocaptured on protein chip arrays is described. The underglycosylation of glycoproteins in CDG-I produces glycoforms of transferrin with masses lower than that of the normal fully glycosylated transferrin. Immobilisation of antitransferrin antibodies on reactive-surface protein chip arrays (RS100) selectively enriched transferrin by at least 100-fold and allowed the detection of patterns of transferrin glycoforms by SELDI-TOF-MS using approximately 0.3 microL of serum/plasma. Abnormal patterns of immunocaptured transferrin were detected in patients with known defects in glycosylation (CDG-Ia, CDG-Ib, CDG-Ic, CDG-If and CDG-Ih) and in patients in whom the basic defect has not yet been identified (CDG-Ix). The correction of the N-glycosylation defect in a patient with CDG-Ib after mannose therapy was readily detected. A patient who had an abnormal transferrin profile by IEF but a normal profile by SELDI-TOF-MS analysis was shown to have an amino acid polymorphism by sequencing transferrin by quadrupole-TOF MS. Complete agreement was obtained between analysis of immunocaptured transferrin by SELDI-TOF-MS and the IEF profile of transferrin, the clinical severity of the disease and the levels of aspartylglucosaminidase activity (a surrogate marker for the diagnosis of CDG-I). SELDI-TOF-MS of transferrin immunocaptured on protein chip arrays is a highly sensitive diagnostic method for CDG-I, which could be fully automated using microtitre plates and robotics.     

Comparative serum glycoproteomics using lectin selected sialic acid glycoproteins with mass spectrometric analysis: application to pancreatic cancer serum

A strategy is developed in this study for identifying sialylated glycoprotein markers in human cancer serum. This method consists of three steps: lectin affinity selection, a liquid separation and characterization of the glycoprotein markers using mass spectrometry. In this work, we use three different lectins (Wheat Germ Agglutinin, (WGA) Elderberry lectin,(SNA), Maackia amurensis lectin, (MAL)) to extract sialylated glycoproteins from normal and cancer serum. Twelve highly abundant proteins are depleted from the serum using an IgY-12 antibody column. The use of the different lectin columns allows one to monitor the distribution of alpha(2,3) and alpha(2,6) linkage type sialylation in cancer serum vs that in normal samples. Extracted glycoproteins are fractionated using NPS-RP-HPLC followed by SDS-PAGE. Target glycoproteins are characterized further using mass spectrometry to elucidate the carbohydrate structure and glycosylation site. We applied this approach to the analysis of sialylated glycoproteins in pancreatic cancer serum. Approximately 130 sialylated glycoproteins are identified using microLC-MS/MS. Sialylated plasma protease C1 inhibitor is identified to be down-regulated in cancer serum. Changes in glycosylation sites in cancer serum are also observed by glycopeptide mapping using microLC-ESI-TOF-MS where the N83 glycosylation of alpha1-antitrypsin is down regulated. In addition, the glycan structures of the altered proteins are assigned using MALDI-QIT-MS. This strategy offers the ability to quantitatively analyze changes in glycoprotein abundance and detect the extent of glycosylation alteration as well as the carbohydrate structure that correlate with cancer.     

An oligosaccharyltransferase from Leishmania major increases the N‐glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana

N‐glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central‐protein complex facilitating the N‐glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N‐glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single‐subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well‐established production platform for recombinant proteins. A fluorescent protein‐tagged LmSTT3D variant was predominately found in the ER and co‐located with plant oligosaccharyltransferase subunits. Co‐expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N‐glycosylation site occupancy on all N‐glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N‐glycosylation efficiency in plants.     

Congenital Disorders of Glycosylation: CDG-I, CDG-II, and beyond

The Congenital Disorders of Glycosylation (CDG) are a collection of over 20 inherited diseases that impair protein N-glycosylation. The clinical appearance of CDG patients is quite diverse making it difficult for physicians to recognize them. A simple blood test of transferrin glycosylation status signals a glycosylation abnormality, but not the specific defect. An abnormal trasferrin glycosylation pattern suggests that the defect is in either genes that synthesize and add the precursor glycan (Glc(3)Man(9)GlcNAc(2)) to proteins (Type I) or genes that process the protein-bound N-glycans (Type II). Type I defects create unoccupied glycosylation sites, while Type II defects give fully occupied sites with abnormally processed glycans. These types are expected to be mutually exclusive, but a group of patients is now emerging who have variable coagulopathy and hypoglycemia together with a combination of Type I and Type II transferrin features. This surprising finding makes identifying their defects more challenging, but the defects and associated clinical manifestations of these patients suggest that the N-glycosylation pathway has some secrets left to share.     

DDOST mutations identified by whole-exome sequencing are implicated in congenital disorders of glycosylation

Congenital disorders of glycosylation (CDG) are inherited autosomal-recessive diseases that impair N-glycosylation. Approximately 20% of patients do not survive beyond the age of 5 years old as a result of widespread organ dysfunction. Although most patients receive a CDG diagnosis based on abnormal glycosylation of transferrin, this test cannot provide a genetic diagnosis; indeed, many patients with abnormal transferrin do not have mutations in any known CDG genes. Here, we combined biochemical analysis with whole-exome sequencing (WES) to identify the genetic defect in an untyped CDG patient, and we found a 22 bp deletion and a missense mutation in DDOST, whose product is a component of the oligosaccharyltransferase complex that transfers the glycan chain from a lipid carrier to nascent proteins in the endoplasmic reticulum lumen. Biochemical analysis with three biomarkers revealed that N-glycosylation was decreased in the patient's fibroblasts. Complementation with wild-type-DDOST cDNA in patient fibroblasts restored glycosylation, indicating that the mutations were pathological. Our results highlight the power of combining WES and biochemical studies, including a glyco-complementation system, for identifying and confirming the defective gene in an untyped CDG patient. This approach will be very useful for uncovering other types of CDG as well.     

Mass spectrometry for congenital disorders of glycosylation, CDG

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.     

Mapping N-linked glycosylation sites in the secretome and whole cells of Aspergillus niger using hydrazide chemistry and mass spectrometry

Protein glycosylation (e.g., N-linked glycosylation) is known to play an essential role in both cellular functions and secretory pathways; however, our knowledge of in vivo N-glycosylated sites is very limited for the majority of fungal organisms including Aspergillus niger. Herein, we present the first extensive mapping of N-glycosylated sites in A. niger by applying an optimized solid phase glycopeptide enrichment protocol using hydrazide-modified magnetic beads. The enrichment protocol was initially optimized using both mouse blood plasma and A. niger secretome samples, and it was demonstrated that the protein-level enrichment protocol offered superior performance over the peptide-level protocol. The optimized protocol was then applied to profile N-glycosylated sites from both the secretome and whole cell lysates of A. niger. A total of 847 N-glycosylated sites from 330 N-glycoproteins (156 proteins from the secretome and 279 proteins from whole cells) were confidently identified by LC-MS/MS. The identified N-glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, Golgi apparatus, lysosome, and storage vacuoles, supporting the important role of N-glycosylation in the secretory pathways. In addition, these glycoproteins are involved in many biological processes including gene regulation, signal transduction, protein folding and assembly, protein modification, and carbohydrate metabolism. The extensive coverage of N-glycosylated sites and the observation of partial glycan occupancy on specific sites in a number of enzymes provide important initial information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.     

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.     

Biosynthesis of abnormally glycosylated hepatoma secretory proteins in cell cultures

We studied, by electrophoretic techniques, the physiochemical properties of 4 glycoproteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein and transferrin synthesized by three different human hepatoma cell lines. A common feature was the export of glycoproteins with retarded electrophoretic mobility, indicating incomplete sialylation, and a predominance of atypical, highly branched carbohydrate chains. The abnormal glycosylation pattern may be specific for malignant transformation of hepatocytes and possibly related to the intracellular accumulation of some of these proteins in malignant cells.