Linkage-Specific Sialic Acid Derivatization for MALDI-TOF-MS Profiling of IgG Glycopeptides

Abstract

Glycosylation is a common co- and post-translational protein modification, having a large influence on protein properties like conformation and solubility. Furthermore, glycosylation is an important determinant of efficacy and clearance of biopharmaceuticals such as immunoglobulin G (IgG). Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) shows potential for the site-specific glycosylation analysis of IgG at the glycopeptide level. With this approach, however, important information about glycopeptide sialylation is not duly covered because of in-source and metastable decay of the sialylated species. Here, we present a highly repeatable sialic acid derivatization method to allow subclass-specific MALDI-TOF-MS analysis of tryptic IgG glycopeptides. The method, employing dimethylamidation with the carboxylic acid activator 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC) and the catalyst 1-hydroxybenzotriazole (HOBt), results in different masses for the functionally divergent α2,3- and α2,6-linked sialic acids. Respective lactonization and dimethylamidation leads to their direct discrimination in MS and importantly, both glycan and peptide moieties reacted in a controlled manner. In addition, stabilization allowed the acquisition of fragmentation spectra informative with respect to glycosylation and peptide sequence. This was in contrast to fragmentation spectra of underivatized samples, which were dominated by sialic acid loss. The method allowed the facile discrimination and relative quantitation of IgG Fc sialylation in therapeutic IgG samples. The method has considerable potential for future site- and sialic acid linkage-specific glycosylation profiling of therapeutic antibodies, as well as for subclass-specific biomarker discovery in clinical IgG samples derived from plasma.

Changes in Healthy Human IgG Fc-Glycosylation after Birth and during Early Childhood

“…This was accomplished by a previously published, rapid and robust MALDI-TOF-MS workflow in 96-well plate format. 23 Although various studies describing IgG Fc-glycosylation in both healthy and diseased individuals have already been performed 3,6,13,[16][17][18][19] , this is the first study that takes very young children (age 0.1 to 3.9) into account and compares them to newborns. It is also the first study describing Fc-glycosylation during childhood in a subclass-specific way, giving a full and specific characterization of IgG1 Fc-glycosylation and specific characterization of changes in galactosylation and sialylation of IgG2/3.…”

“…These values were slightly higher than the CV of 2.2% reported previously for the most abundant glycopeptide in isolated IgG1 measured 12 times in the same samples, using this method. 23 However, in the method reported before, the isolation of IgG from plasma was not taken into account.…”

“…We analyzed IgG Fc-glycopeptides using a previously reported matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) method. 23 The method includes the derivatization of the IgG glycopeptides to stabilize the sialylated species during MALDI ionization and distinguishes α2,3-and α2,6-linked sialic acids by forming a lactone (-18.011 Da) or a dimethylamide (+27.047 Da), respectively. This characterization revealed that when children start to produce their own IgG, about one month after birth 24 , their galactosylation, sialylation and bisection decreases and their fucosylation increases compared to IgG found in umbilical cord samples that represent the IgG of both the mothers and the newborns.…”

Gangliosides of the Vertebrate Nervous System

“…The biological significance of these distributions has yet to be understood. Despite its power, MALDI-MS mapping is prone to in-source loss of sialic acids, which are differentially sensitive to the experimental conditions [105]. This can lead to under-representation of major brain gangliosides GT1b, GD1b, and GD1a and over-representation of GM1, for example.…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…Furthermore, recent studies have explored the feasibility of measuring deprotonated glycopeptides in negative ion mode with (130) and without (131) derivatization. Innovative strategies for the derivatization of sialic acid residues have also been designed to achieve linkage-specific (␣2,3 versus ␣2,6-sialyl) information at the glycopeptide level (132) and to obtain more equal ionization response from neutral and sialylated glycopeptides (133). Furthermore, attempts to generate universal workflows using Pronase digestion and C18-PGC LC-MS/MS instead of the conventional RP-LC-MS/MS of tryptic glycopeptides were described (39,134).…”