Characterization of antithrombin isoforms and latent forms by ion exchange chromatography coupled to mass spectrometry.

Antithrombin is a key protein of the coagulation system belonging to the serine protease inhibitor family. Antithrombin preparations are used as a therapeutic treatment for patients with decreased antithrombin activity. Elucidating the structural features of this protein is an important part of the control strategy to assure a high quality. This study presents an ion exchange chromatographic method coupled to mass spectrometry capable of characterizing antithrombin post-translational modifications such as N-glycosylation, phosphorylation or deamidation. Furthermore, the method was successfully used to evidence irreversible/inactive conformers of antithrombin which are commonly observed for serine protease inhibitors and referred to as latent forms.

C-terminal lysine clipping of IgG1: impact on binding to human FcγRIIIa and neonatal Fc receptors.

Monoclonal antibodies (mAbs) display numerous structural attributes, some of them may impact their safety and/or efficacy profiles. C-terminal lysine clipping is a common phenomenon occurring during the bioproduction of mAbs and leads to variable amounts of final process-related charge variants. If Fc-glycosylation has been by far the most documented critical quality attribute (CQA), the potential impacts of mAb C-terminal lysine content is far less reported, particularly on the ability of these basic variants to bind human Fc receptors. To address this question, three charge variant species having zero (K0), one (K1) and two (K2) C-terminal lysine(s) were isolated with high purity from an in-house human IgG1 by preparative strong-cation exchange (SCX) chromatography. A comprehensive biophysical characterization of these three fractions was undertaken, demonstrating their high similarity in terms of structural homogeneity, with a particular attention paid on their respective N-glycosylation profiles. The binding affinity of the fractions to human FcγRIIIa-Val176 was assessed both by affinity chromatography and surface plasmon resonance (SPR), and to human neonatal Fc receptor (FcRn) by affinity chromatography. Results demonstrate that the three charge variants did not show any significant binding difference for the two tested human Fc receptors, translating certainly to comparable biological properties. As a consequence, C-terminal lysine clipping of the present therapeutic IgG1 should not impact both FcRn-dependent pharmacokinetic profiles and FcγRIIIa-driven cytotoxic activities. The methods used in this study can be widely applied to other IgG1 to define criticality of the C-terminal lysine clipping as a CQA.

Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls.

Despite significant analytical improvements during this last decade, characterizing the whole integrity of monoclonal antibodies during their bioproduction remains a challenge. In this study, we report a new analytical approach to evaluate the overall heterogeneity/integrity of mAbs by LC-MS after combined proteolysis at their lower- and upper-hinge sites using the immunoglobulin-degrading enzymes IdeS and IgdE respectively. The whole sample preparation did not use any harsh conditions such as low pH, high temperature or reductive conditions and enables the splitting of mAbs structure into three fragments, namely the hinge dimer, Fab and Fc/2. Using the NIST mAb reference material, this method was demonstrated to be particularly suited for the analysis of mAbs disulfide bridges. The three fragments as well as their corresponding free sulfhydryl forms were well separated by chromatography and identified online by mass spectrometry. The method was then successfully applied to several mAbs of variable hydrophobicities.

Mass spectrometry based analysis of human plasma-derived factor X revealed novel post-translational modifications.

Human coagulation factor X is a central component of the blood coagulation cascade that converts, under its activated form, prothrombin into thrombin. Generation of thrombin is the final step of the clotting cascade that leads to the clot by polymerization of fibrinogen molecules into a fibrin network. Today, research of new by-passing agents of the coagulation may contribute to an increased interest for human factor X, which may, in consequence, lead to the need of a more exhaustive picture of its structural features. Several post-translational modifications of human factor X such as γ-carboxylation/ß-hydroxylation of the N-terminal light chain and N-/O-glycosylation of the activation peptide have been described. But, so far as we know, no comprehensive studies of its post-translational modifications have been reported. In this article we report an exhaustive structural analysis of human factor X by mass spectrometry using successive protein and peptide mapping. Surprisingly, human factor X was found to be mostly O-glucosylated on its light chain at Ser106 position, Ser9 of its activation peptide is phosphorylated at about 30% and its C-terminal heavy chain is fully O-glycosylated at Thr249 by a mucin-type O-glycan (HexNAc-Hex-NeuAc). The knowledge of these post-translational modifications is mandatory for the development of recombinant molecules.

Site-specific N-glycosylation analysis of human factor XI: Identification of a noncanonical NXC glycosite.

Human factor XI (hFXI) is a 160-kDa disulphide-linked homodimer zymogen involved in the coagulation cascade. Its deficiency results in bleeding diathesis referred to as hemophilia C. hFXI bears five N-glycosylation consensus sites per monomer, N72 , N108 , N335 on the heavy chain and N432 , N473 on the light chain. This study reports the first in-depth glycosylation analysis of hFXI based on advanced MS approaches. Hydrophilic interaction LC and MS characterization and quantification of the N-glycans showed that the two major forms are complex biantennary mono-α2,6-sialylated (A2 S1 , 20%) and bis-α2,6-sialylated structures (A2 S2 , 66%). Minor triantennary structures (A3 S3 F, ∼1.5%; A3 S3 , ∼2%) were also identified. MS analyses of intact hFXI revealed full occupation of two of the three heavy-chain glycosites and almost full-site occupancy of the light chain. Analysis of hFXI glycopeptides by LC-MS/MS enabled site-specific glycan profiling and occupancy. It was evidenced that N335 was not glycosylated and that N72 and N108 were fully occupied, whereas N432 and N473 were occupied at about 92 and 95%, respectively. We also identified a new glycosite of the noncanonical format NXC at N145 , occupied at around 5%. These data provide valuable structural information useful to understand the potential roles of N-glycosylation on hFXI function and could serve as a structural reference.

N-/O-glycosylation analysis of human FVIIa produced in the milk of transgenic rabbits.

Human coagulation factor VIIa is a glycoprotein that promotes haemostasis through activation of the coagulation cascade extrinsic pathway. Most haemophilia A/B patients with inhibitors are treated by injection of plasma-derived or recombinant FVIIa. The use of recombinant products raises questions about the ability of the host cell to produce efficiently post-translationally modified proteins. Glycosylation is especially critical considering that it can modulate protein safety and efficacy. The present paper reports the N-/O-glycosylation pattern of a new recombinant human factor VIIa expressed in the mammary glands of transgenic rabbits. Glycosylation was investigated by chromatography and advanced mass spectrometry techniques for glycan identification and quantitation. Mass spectrometry (MS)/MS analyses were performed to confirm the glycan structures as well as the position and branching of specific monosaccharides or substituents. The two N-glycosylation sites were found to be fully occupied mostly by mono- and bi-sialylated biantennary complex-type structures, the major form being A(2)G(2)S(1). Some oligomannose/hybrid structures were retrieved in lower abundance, the major ones being GlcNAcα1,O-phosphorylated at the C6-position of a Man residue (Man-6-(GlcNAcα1,O-)phosphate motif) as commonly observed on lysosomal proteins. No immunogenic glycotopes such as Galili (Galα1,3Gal) and HD antigens (N-glycolylneuraminic acid (NeuGc)) were detected. Concerning O-glycosylation, the product exhibited O-fucose and O-glucose-(xylose)(0, 1, 2) motifs as expected. The N-glycosylation consistency was also investigated by varying production parameters such as the period of lactation, the number of consecutive lactations and rabbit generations. Results show that the transgenesis technology is suitable for the long-term production of rhFVIIa with a reproducible glycosylation pattern.

Glycosylation pattern of mature dimeric leukocyte and recombinant monomeric myeloperoxidase: glycosylation is required for optimal enzymatic activity.

The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro- and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and the three-dimensional structure of MPO.

Analysis of N- and O-linked glycans from glycoproteins using MALDI-TOF mass spectrometry.

Glycosylation represents the most common of all known protein post-translational modifications. Carbohydrates can modulate the biological functions of a glycoprotein, protect a protein against hydrolysis via protease activity, and reduce or prevent aggregation of a protein. The determination of the carbohydrate structure and function in glycoproteins remains one of the most challenging tasks given to biochemists, as these molecules can exhibit complex branched structures that can differ in linkage and in the level of branching. In this review, we will present the approach followed in our laboratory for the elucidation of N- and O-glycan chains of glycoproteins. First, reduced/carboxamidomethylated glycoproteins are digested with a protease or a chemical reagent. N-Glycans are then released from the resulting peptides/glycopeptides via digestion with peptide N-glycosidase F (PNGase F). Oligosaccharides released by PNGase F are separated from peptides and glycopeptides using a C18 Sep-Pak, and their methylated derivatives are characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). O-Glycans are released by reductive elimination, which are permethylated, purified on a Sep-Pak C18 cartridge, and analyzed with MALDI-TOF-MS. Finally, to confirm the structures N-glycans released by PNGase F are characterized using MALDI-TOF-MS following on-plate sequential exoglycosidase digestions. The clean-up procedures of native and permethylated oligosaccharides for an efficient MALDI-TOF-MS analysis will also be described. This strategy was applied to calf fetuin and glycoproteins present in human serum.

A mass spectrometric strategy for profiling glycoproteinoses, Pompe disease, and sialic acid storage diseases.

Glycoproteinoses, Pompe disease, and sialic acid storage diseases are characterized by a massive accumulation of unprocessed oligosaccharides and/or glycoconjugates in urine. The identification of these glycocompounds is essential for a proper diagnosis. In this study, we investigated the potential of MALDI-TOF-MS to identify glycocompounds present in urine from patients with different inborn errors of glycan metabolism. Urinary glycocompounds were permethylated, and analyzed using GC-MS and MALDI-TOF-MS. In order to confirm tentative assignments, a second aliquot of urine was purified on a C18 Sep-Pak cartridge and glycocompounds were desalted on a column of nonporous graphitized carbon. The glycocompounds were then sequentially on-plate digested using an array of exoglycosidases. A range of disease-specific oligosaccharides as well as glycopeptides was identified for all oligosacchariduria models. In addition, free sialic acid accumulated in urine from a patient suffering from French-type sialuria, has been detected by a GC-MS approach, which could be applied to other sialic acid storage diseases. This procedure is simple, and can be performed in few simple steps in less than 24 h. This current method can be applied for newborn screening for other inherited metabolic diseases as well as for assessing treatments in clinical trials.

A rapid mass spectrometric strategy for the characterization of N- and O-glycan chains in the diagnosis of defects in glycan biosynthesis.

Glycosylation of proteins is a very complex process which involves numerous factors such as enzymes or transporters. A defect in one of these factors in glycan biosynthetic pathways leads to dramatic disorders named congenital disorders of glycosylation (CDG). CDG can affect the biosynthesis of not only protein N-glycans but also O-glycans. The structural analysis of glycans on serum glycoproteins is essential to solving the defect. For this reason, we propose in this paper a strategy for the simultaneous characterization of both N- and O-glycan chains isolated from the serum glycoproteins. The serum (20 microL) is used for the characterization of N-glycans which are released by enzymatic digestion with PNGase F. O-glycans are chemically released by reductive elimination from whole serum glycoproteins using 10 microL of the serum. Using strategies based on mass spectrometric analysis, the structures of N- and O-glycan chains are defined. These strategies were applied on the sera from one patient with CDG type IIa, and one patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex.

The use of mass spectrometry for the proteomic analysis of glycosylation.

Of all protein PTMs, glycosylation is by far the most common, and is a target for proteomic research. Glycosylation plays key roles in controlling various cellular processes and the modifications of the glycan structures in diseases highlight the clinical importance of this PTM. Glycosylation analysis remains a difficult task. MS, in combination with modern separation methodologies, is one of the most powerful and versatile techniques for the structural analysis of glycoconjugates. This review describes methodologies based on MS for detailed characterization of glycoconjugates in complex biological samples at the sensitivity required for proteomic work.

Site-specific glycosylation analysis of the bovine lysosomal alpha-mannosidase.

Lysosomal alpha-mannosidase is a broad specificity exoglycosidase involved in the ordered degradation of glycoproteins. The bovine enzyme is used as an important model for understanding the inborn lysosomal storage disorder alpha-mannosidosis. This enzyme of about 1,000 amino acids consists of five peptide chains, namely a- to e-peptides and contains eight N-glycosylation sites. The N(497) glycosylation site of the c-peptide chain is evolutionary conserved among LAMANs and is very important for the maintenance of the lysosomal stability of the enzyme. In this work, relying on an approach based on mass spectrometric techniques in combination with exoglycosidase digestions and chemical derivatizations, we will report the detailed structures of the N-glycans and their distribution within six of the eight N-glycosylation sites of the bovine glycoprotein. The analysis of the PNGase F-released glycans from the bovine LAMAN revealed that the major structures fall into three classes, namely high-mannose-type (Fuc(0-1)Glc(0-1)Man(4-9)GlcNAc(2)), hybrid-type (Gal(0-1)Man(4-5)GlcNAc(4)), and complex-type (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(3-5)) N-glycans, with core fucosylation and bisecting GlcNAc. To investigate the exact structure of the N-glycans at each glycosylation site, the peptide chains of the bovine LAMAN were separated using SDS-PAGE and in-gel deglycosylation. These experiments revealed that the N(497) and N(930) sites, from the c- and e-peptides, contain only high-mannose-type glycans Glc(0-1)Man(5-9)GlcNAc(2), including the evolutionary conserved Glc(1)Man(9)GlcNAc(2) glycan, and Fuc(0-1)Man(3-5)GlcNAc(2), respectively. Therefore, to determine the microheterogeneity within the remaining glycosylation sites, the glycoprotein was reduced, carboxymethylated, and digested with trypsin. The tryptic fragments were then subjected to concanavalin A (Con A) affinity chromatography, and the material bound by Con A-Sepharose was purified using reverse-phase high-performance liquid chromatography (HPLC). The tandem mass spectrometry (ESI-MS/MS) and the MALDI analysis of the PNGase F-digested glycopeptides indicated that (1) N(692) and N(766) sites from the d-peptide chain both bear glycans consisting of high-mannose (Fuc(0-1)Man(3-7)GlcNAc(2)), hybrid (Fuc(0-1) Gal(0-1)Man(4-5)GlcNAc(4)), and complex (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(4-5)) structures; and (2) the N(367) site, from the b-peptide chain, is glycosylated only with high-mannose structures (Fuc(0-1)Man(3-5)GlcNAc(2)). Taking into consideration the data obtained from the analysis of either the in-gel-released glycans from the abc- and c-peptides or the tryptic glycopeptide containing the N(367) site, the N(133) site, from the a-peptide, was shown to be glycosylated with truncated and high-mannose-type (Fuc(0-1)Man(4-5)GlcNAc(2)), complex-type (Fuc(0-1)Gal(0-1)Man(3)GlcNAc(5)), and hybrid-type (Fuc(0-1)Gal(0-1)Man(5)GlcNAc(4)) glycans.

Structural analysis of permethylated oligosaccharides using electrospray ionization quadrupole time-of-flight tandem mass spectrometry and deutero-reduction.

Deutero-reduced permethylated oligosaccharides were analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) using a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, fitted with a nanoflow ESI source. Under these ionization conditions such derivatives preferentially form sodiated molecular species in addition to protonated molecular species. Under collision-induced dissociation, protonated and sodiated molecular species yield simple and predictable fragment mass spectra. A systematic study was conducted on a series of deutero-reduced permethylated glycans to allow rationalization of the fragmentation processes. MS/MS spectra were characterized by fragments resulting from the cleavage of glycosidic bonds. These fragments originating from both the reducing and the non-reducing ends of the glycan yield information on sequence and branching. Furthermore, the substituent 3-linked to a HexNAc unit was readily eliminated. Special attention was devoted to a systematic study of fucosylated glycans. The fucosylated deutero-reduced permethylated glycans were submitted to an acidic hydrolysis, releasing specifically the fucosyl residues. The nascent free hydroxyl groups were subsequently CD3-labelled in order to determine the positions initially bearing the fucosyl residues along the oligosaccharide backbone. This methodology was finally applied to characterize a glycan pool enzymatically released from glycoproteins. The present data show that structural elucidation can be achieved at the 50 fmol level.