Valegh Faid

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.

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.

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 A2G2S1. 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.

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.

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 (A2S1, 20%) and bis‐α2,6‐sialylated structures (A2S2, 66%). Minor triantennary structures (A3S3F, ∼1.5%; A3S3, ∼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.

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

HighlightsAnalysis of mAbs using combined IgdE and IdeS hinge specific proteolysis.IgdE cleaves mAbs at their upper hinge region in non‐reducing conditions.Characterization of mAbs free sulfhydryls and other PTMs. ABSTRACT 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/β‐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.