Maximizing Glycoproteomics Results through an Integrated Parallel Accumulation Serial Fragmentation Workflow.

Glycoproteins play important roles in numerous physiological processes and are often implicated in disease. Analysis of site-specific protein glycobiology through glycoproteomics has evolved rapidly in recent years thanks to hardware and software innovations. Particularly, the introduction of parallel accumulation serial fragmentation (PASEF) on hybrid trapped ion mobility time-of-flight mass spectrometry instruments combined deep proteome sequencing with separation of (near-)isobaric precursor ions or converging isotope envelopes through ion mobility separation. However, the reported use of PASEF in integrated glycoproteomics workflows to comprehensively capture the glycoproteome is still limited. To this end, we developed an integrated methodology using timsTOF Pro 2 to enhance N-glycopeptide identifications in complex mixtures. We systematically optimized the ion optics tuning, collision energies, mobility isolation width, and the use of dopant-enriched nitrogen gas (DEN). Thus, we obtained a marked increase in unique glycopeptide identification rates compared to standard proteomics settings, showcasing our results on a large set of glycopeptides. With short liquid chromatography gradients of 30 min, we increased the number of unique N-glycopeptide identifications in human plasma samples from around 100 identifications under standard proteomics conditions to up to 1500 with our optimized glycoproteomics approach, highlighting the need for tailored optimizations to obtain comprehensive data.

N-linked glycosylation of the M-protein variable region: glycoproteogenomics reveals a new layer of personalized complexity in multiple myeloma.

OBJECTIVES: Multiple myeloma (MM) is a plasma cell malignancy characterized by a monoclonal expansion of plasma cells that secrete a characteristic M-protein. This M-protein is crucial for diagnosis and monitoring of MM in the blood of patients. Recent evidence has emerged suggesting that N-glycosylation of the M-protein variable (Fab) region contributes to M-protein pathogenicity, and that it is a risk factor for disease progression of plasma cell disorders. Current methodologies lack the specificity to provide a site-specific glycoprofile of the Fab regions of M-proteins. Here, we introduce a novel glycoproteogenomics method that allows detailed M-protein glycoprofiling by integrating patient specific Fab region sequences (genomics) with glycoprofiling by glycoproteomics. METHODS: Glycoproteogenomics was used for the detailed analysis of de novo N-glycosylation sites of M-proteins. First, Genomic analysis of the M-protein variable region was used to identify de novo N-glycosylation sites. Subsequently glycopeptide analysis with LC-MS/MS was used for detailed analysis of the M-protein glycan sites. RESULTS: Genomic analysis uncovered a more than two-fold increase in the Fab Light Chain N-glycosylation of M-proteins of patients with Multiple Myeloma compared to Fab Light Chain N-glycosylation of polyclonal antibodies from healthy individuals. Subsequent glycoproteogenomics analysis of 41 patients enrolled in the IFM 2009 clinical trial revealed that the majority of the Fab N-glycosylation sites were fully occupied with complex type glycans, distinguishable from Fc region glycans due to high levels of sialylation, fucosylation and bisecting structures. CONCLUSIONS: Together, glycoproteogenomics is a powerful tool to study de novo Fab N-glycosylation in plasma cell dyscrasias.

Glycoproteomics in Cerebrospinal Fluid Reveals Brain-Specific Glycosylation Changes.

The glycosylation of proteins plays an important role in neurological development and disease. Glycoproteomic studies on cerebrospinal fluid (CSF) are a valuable tool to gain insight into brain glycosylation and its changes in disease. However, it is important to consider that most proteins in CSFs originate from the blood and enter the CSF across the blood-CSF barrier, thus not reflecting the glycosylation status of the brain. Here, we apply a glycoproteomics method to human CSF, focusing on differences between brain- and blood-derived proteins. To facilitate the analysis of the glycan site occupancy, we refrain from glycopeptide enrichment. In healthy individuals, we describe the presence of heterogeneous brain-type N-glycans on prostaglandin H2-D isomerase alongside the dominant plasma-type N-glycans for proteins such as transferrin or haptoglobin, showing the tissue specificity of protein glycosylation. We apply our methodology to patients diagnosed with various genetic glycosylation disorders who have neurological impairments. In patients with severe glycosylation alterations, we observe that heavily truncated glycans and a complete loss of glycans are more pronounced in brain-derived proteins. We speculate that a similar effect can be observed in other neurological diseases where a focus on brain-derived proteins in the CSF could be similarly beneficial to gain insight into disease-related changes.

Loss of the AMD-associated B3GLCT gene affects glycosylation of TSP1 without impairing secretion in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) has been associated with protective genetic variants in the ß1-3 glucosyltransferase (B3GLCT) locus through genome-wide association studies. B3GLCT mediates modification of proteins with thrombospondin type I repeats (TSR) that contain O-linked glucose ß1-3 fucose and C-linked mannose glycosylation motifs. B3GLCT-mediated modification is required for proper secretion of TSR-containing proteins. We aimed to start understanding the role of B3GLCT in AMD by evaluating its effect on glycosylation and secretion of proteins from retinal pigment epithelium (RPE) cells. We generated B3GLCT knockout (KO) RPE cells and analyzed glycosylation and secretion of thrombospondin 1 (TSP1), a protein involved in cellular processes highly relevant to AMD. Glycopeptide analysis confirmed the presence of the glucose-ß1,3-fucose product of B3GLCT on TSP1 in wildtype (WT) cells and its absence in KO cells. C-mannosylation was variably present on WT TSP1 and increased on TSR domains 1 and 3 in KO cells. Secretion of TSP1 was not affected by the absence of B3GLCT, even not when TSP1 was upregulated by TNFα treatment or when TSP1 was overexpressed in HEK293T cells. Future research is needed to elucidate the effect of the observed glycosylation defects in the context of AMD, which might involve functional loss of TSP1 or effects on other TSR proteins.

Active site variants in STT3A cause a dominant type I congenital disorder of glycosylation with neuromusculoskeletal findings.

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs.

Congenital disorder of glycosylation caused by starting site-specific variant in syntaxin-5.

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein syntaxin-5 (Stx5) is essential for Golgi transport. In humans, the STX5 mRNA encodes two protein isoforms, Stx5 Long (Stx5L) from the first starting methionine and Stx5 Short (Stx5S) from an alternative starting methionine at position 55. In this study, we identify a human disorder caused by a single missense substitution in the second starting methionine (p.M55V), resulting in complete loss of the short isoform. Patients suffer from an early fatal multisystem disease, including severe liver disease, skeletal abnormalities and abnormal glycosylation. Primary human dermal fibroblasts isolated from these patients show defective glycosylation, altered Golgi morphology as measured by electron microscopy, mislocalization of glycosyltransferases, and compromised ER-Golgi trafficking. Measurements of cognate binding SNAREs, based on biotin-synchronizable forms of Stx5 (the RUSH system) and Förster resonance energy transfer (FRET), revealed that the short isoform of Stx5 is essential for intra-Golgi transport. Alternative starting codons of Stx5 are thus linked to human disease, demonstrating that the site of translation initiation is an important new layer of regulating protein trafficking.

Dissecting Total Plasma and Protein-Specific Glycosylation Profiles in Congenital Disorders of Glycosylation.

Protein N-glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in protein N-glycan biosynthesis. Here, we present insights into the disrupted N-glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking. We studied enzymatically released N-glycans of total plasma proteins and affinity purified immunoglobulin G (IgG) from patients and healthy controls using mass spectrometry (MS). The applied method allowed the differentiation of sialic acid linkage isomers via their derivatization. Furthermore, protein-specific glycan profiles were quantified for transferrin and IgG Fc using electrospray ionization MS of intact proteins and glycopeptides, respectively. Next to the previously described glycomic effects, we report unprecedented sialic linkage-specific effects. Defects in proteins involved in Golgi trafficking (COG5-CDG) and CMP-sialic acid transport (SLC35A1-CDG) resulted in lower levels of sialylated structures on plasma proteins as compared to healthy controls. Findings for these specific CDGs include a more pronounced effect for α2,3-sialylation than for α2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation.

Biallelic variants in SLC35C1 as a cause of isolated short stature with intellectual disability.

Variants in SLC35C1 underlie leucocyte adhesion deficiency (LADII) or congenital disorder of glycosylation type 2c (CDGIIc), an autosomal recessive disorder of fucosylation. This immunodeficiency syndrome is generally characterized by severe recurrent infections, Bombay blood group, reduced growth and intellectual disability (ID). Features are all caused by an inability to generate key fucosylated molecules due to a defective transport of GDP-fucose into the Golgi. Here we report the use of exome sequencing to identify biallelic variants in SLC35C1 (c.501_503delCTT, p.(Phe168del) and c.891T > G, p.(Asn297Lys)) in an individual with short stature and ID. Retrospective clinical examination based on the genetic findings revealed increased otitis media as the only immunological feature present in this child. Biochemical analysis of patient serum identified a clear but mild decrease in protein fucosylation. Modelling all described missense mutations on a SLC35C1 protein model showed pathogenic substitutions localise to close to the dimer interface, providing insight into the possible pathophysiology of non-synonymous causative variants identified in patients. Our evidence confirms this is the second family presenting with only a subset of features and broadens the clinical presentation of this syndrome. Of note, both families segregated a common allele (p.Phe168del), suggesting there could be an associated genotype-phenotype relationship for specific variants. Based on two out of 14 reported families not presenting with the characteristic features of SLC35C1-CDG, we suggest there is clinical utility in considering this gene in patients with short stature and ID.

In Silico Evaluation of the Binding Site of Fucosyltransferase†8 and First Attempts to Synthesize an Inhibitor with Drug-Like Properties.

Core fucosylation of N-glycans is catalyzed by fucosyltransferase 8 and is associated with various types of cancer. Most reported fucosyltransferase inhibitors contain non-drug-like features, such as charged groups. New starting points for the development of inhibitors of fucosyltransferase 8 using a fragment-based strategy are presented. Firstly, we discuss the potential of a new putative binding site of fucosyltransferase 8 that, according to a molecular dynamics (MD) simulation, is made accessible by a significant motion of the SH3 domain. This might enable the design of completely new inhibitor types for fucosyltransferase 8. Secondly, we have performed a docking study targeting the donor binding site of fucosyltransferase 8, and this yielded two fragments that were linked and trimmed in silico. The resulting ligand was synthesized. Saturation transfer difference (STD) NMR confirmed binding of the ligand featuring a pyrazole core that mimics the guanine moiety. This ligand represents the first low-molecular-weight compound for the development of inhibitors of fucosyltransferase 8 with drug-like properties.

Simultaneous characterization of SNPs and N-glycans from multiple glycosylation sites of intact ß-2-glycoprotein-1 (B2GP1) by ESI-qTOF-MS.

The highly glycosylated ß-2-glycoprotein-1 (B2GP1), also called apolipoprotein H, is a 50 kDa human plasma protein with four or five N-glycosylation sites. Glycosylation of B2GP1 can impact auto antibody recognition leading to the development of antiphospholipid syndrome (APS), which can result in miscarriages or thrombosis. Next to its glycosylation different genetic variants are known to increase the risk of suffering from APS. Here we show that ESI-q/TOF-MS of intact B2GP1 can be used to analyze genetic variants and glycosylation simultaneously. After enrichment of B2GP1 from 16 different plasma samples and subsequent ESI-MS measurement of the intact protein, we detected five different SNPs in our samples either homozygous or heterozygous. The dominant glycan composition shows four biantennary, fully sialylated glycan structures, with a relative proportion of about 30%. We also detected compositions with one or two triantennary glycan structures in lower amounts and fucosylated species with one or two fucosyl residues. Two of our samples showed an unreported partially occupied fifth glycosylation site presumably arising from the presence of SNP variant S88N. Our method allows a fast determination of genetic variants and glycan compositions of human B2GP1 to be potentially used as diagnostic marker.

Quantitation of Glycopeptides by ESI/MS - size of the peptide part strongly affects the relative proportions and allows discovery of new glycan compositions of Ceruloplasmin.

Significant changes of glycan structures are observed in humans if diseases like cancer, arthritis or inflammation are present. Thus, interest in biomarkers based on glycan structures has rapidly emerged in recent years and monitoring disease specific changes of glycosylation and their quantification is of great interest. Mass spectrometry is most commonly used to characterize and quantify glycopeptides and glycans liberated from the glycoprotein of interest. However, ionization properties of glycopeptides can strongly depend on their composition and can therefore lead to intensities that do not reflect the actual proportions present in the intact glycoprotein. Here we show that an increase in the length of the peptide can lead to a more accurate determination and quantification of the glycans. The four glycosylation sites of human serum ceruloplasmin from 17 different individuals were analyzed using glycopeptides of varying peptide lengths, obtained by action of different proteases and by limited digestion. In most cases, highly sialylated compositions showed an increased relative abundance with increasing peptide length. We observed a relative increase of triantennary glycans of up to a factor of three and, even more, MS peaks corresponding to tetraantennary compositions on ceruloplasmin at glycosite 137N in all 17 samples, which we did not detect using a bottom up approach. The data presented here leads to the conclusion that a middle down - or when possible a top down - approach is favorable for qualitative and quantitative analysis of the glycosylation of glycoproteins.

Intact Human Alpha-Acid Glycoprotein Analyzed by ESI-qTOF-MS: Simultaneous Determination of the Glycan Composition of Multiple Glycosylation Sites.

Alpha-1-acid glycoprotein (AGP) is a highly glycosylated protein from human plasma with five N-type glycosylation sites carrying dominantly higher antennary structures and thus represents a challenging target for characterization of glycan heterogeneity. Here, we show that glycan composition over all five glycosylation sites can be determined quantitatively by ESI-qTOF-MS of the intact glycoprotein in negative ion mode. We find numerous glycan species extending the mass range of the glycoprotein species from 35.0 to 38.5 kDa. The dominant glycan compositions contain tri- and tetraantennary structures on all glycosylation sites. The mass degeneracy of two fucosyl units versus one sialic acid was resolved by treating the sample with sialidase and analyzing the resulting desialylated AGP by electrospray ionization-mass spectrometry in positive ion mode. The pattern of nonsialylated oligosaccharides was used for interpretation of the fully sialylated species using bioinformatics tools. From pooled human plasma, we find 90, 101, and 64 different glycan compositions for genetic variants ORM1*F1, ORM1*S, and ORM2, respectively. Glycan structures carry dominantly between 15 and 16 sialic acids indicating an almost complete termination of all antenae with sialic acid. AGP from human plasma samples of single individuals was analyzed as desialylated glycoproteins and showed variations in fucosylation and in the amount of antennary structures between individuals.

Glycan analysis: scope and limitations of different techniques--a case for integrated use of LC-MS(/MS) and NMR techniques.

The structure of glycans from glycoproteins is highly relevant for their function. We tightly integrate liquid chromatography-mass spectrometry (LC-MS), MS/MS, and nuclear magnetic resonance (NMR) data to achieve a complete characterization of even isobaric glycans differing in only one linkage position or in the substitution in one branch. As example, we analyzed ten desialylated underivatized glycans from bovine fibrinogen. The molecules were separated on a PGC column, and LC-MS data allowed an assignment of the compositions of the glycans. MS/MS data of the same glycans allowed elucidation of sequence and to some extent of branching and linkage. All MS/MS fragmentation methods led to multiple dissociations, resulting in several cases in ambiguous data. The MS/MS data were interpreted both by scientists and automatically by software, and the differential results are compared. Additional data from a tight integration of LC-MS and NMR data resulted in a complete structural characterization of the glycans. The acquisition of simple 1D (1)H NMR data led--in combination with LC-MS and MS/MS data--to an unambiguous assignment of the isobaric glycans. Compounds that were not separated in the chromatography could easily be assigned structurally by applying the 3D cross-correlation (3DCC) technology to arrive at NMR spectra of the pure components-without actually separating them. By applying LC-MS, MS/MS, 1D (1)H NMR, and 3DCC together, one can assign glycan structures from glycoconjugates with high confidence affording only 200 pmol of glycan material.