Noortje de Haan (1), Karli R. Reiding (1), Gerda C. M. Vreeker (1), Markus Haberger (2), Dietmar Reusch (2), David Falck (1), Manfred Wuhrer (1,3)
1) Center for Proteomics and Metabolomics, Leiden University Medical Center (2) Pharma Biotech Development Penzberg, Roche Diagnostics GmbH (3) (3) Division of BioAnalytical Chemistry, VU University
Glycosylation is among the most common co- and post-translational protein modifications, affecting the physiological and biochemical properties of a protein in various ways. High-throughput protein glycosylation analysis may be performed by MALDI-TOF-MS of either released glycans or glycopeptides. This approach, however, biases the analysis of sialylated glycoconjugates by metastable decay and variations in ionization. Here, we present approaches for the linkage-specific sialic acid derivatization of both released glycans and glycopeptides followed by MALDI-TOF-MS analysis. The methods are fast and have an excellent intra- and inter-day repeatability. We envision the use of these methods for the monitoring of protein glycosylation, both in the analysis of biopharmaceuticals and for the detection of glycomic disease biomarkers.
Glycosylation is one of the most common and extensive co- and post-translational protein modifications, having a large influence on protein properties as conformation, solubility and half-life . Not only the glycosylation site occupancy is important for the properties of a conjugate, but also the type of monosaccharides comprising the glycan and their linkages. For example, on various glycoproteins in certain types of cancer, α2,6-linkage of a N-acetylneuraminic acid to a neighboring galactose can promote tumor survival by inhibition of galectin-dependent apoptosis, while α2,3-linkage in a sialyl Lewis motif can assist metastasis by allowing adherence to endothelial selectins [2-5]. Furthermore, a decrease in galactosylation of immunoglobulin G (IgG) glycans is linked to an increase in inflammation in various disease settings [e.g. 6]. Glycosylation is also an important determinant of efficacy and clearance of biopharmaceuticals such as IgG, making careful analysis and control of glycosylation during the production a prerequisite to ensure proper activity .
High-throughput protein glycosylation analysis is performed by (tandem) mass spectrometric analysis of either released glycans or glycopeptides. Analysis of released glycans provides a broad overview of the glycome of a certain complex sample (like plasma or tissue) or of a single isolated protein. On the other hand, glycopeptide analysis enables us to study glycosylation in a protein- and site-specific way, giving information about both the glycan structure and the peptide sequence. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a fast and convenient method for both glycan and glycopeptide analysis. Using this method, however, information on monosaccharide linkages is often difficult to obtain and the analysis of sialylated glycoconjugates is biased by metastable decay and variations in ionization and salt adduction . We here present approaches for the derivatization of the carboxylic acids on both released glycans and glycopeptides, employing the carboxylic acid activator 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC) and catalyst 1-hydroxybenzotriazole (HOBt) in combination with various nucleophiles. By testing a range of reagents and conditions the reactions on released glycans and glycopeptides were optimized to perform in a selective way for differently linked sialic acids, introducing a mass difference between α2,3- and α2,6-linked species. In addition, for the glycopeptide derivatization, reactions were controlled to perform fully selective on the carboxylic acids in the peptide moiety.
To enable the relative quantification of released glycans, they were labeled after sialic acid stabilization using the INLIGHT™ tagging kit. This kit contains two (phenetylphenyl)acetohydrazide labels, of which one is isotopically labeled with six 13C atoms in the phenetyl ring.
Two highly repeatable sialic acid derivatization methods were developed allowing MALDI-TOF-MS detection of both released glycans and glycopeptides in a sialic acid linkage-specific manner. Released glycans were derivatized by a very mild and facile reaction with ethanol in the presence of EDC and HOBt . This reaction induces ethylation of α2,6-linked sialic acids, whereas at the same time α2,3-linked sialic acids lose water by forming a lactone involving the neighboring galactose. Using this protocol, sialic acids were stabilized and a mass difference of 46.04 Da between α2,3- and α2,6-linked sialic acids was introduced. In highly complex samples like human plasma, more than 100 different glycans could be detected by MALDI-TOF-MS after the derivatization. In addition, highly informative fragmentation spectra of both sialylated and non-sialylated species were obtained. Labeling of the esterified glycan samples with the heavy and the light INLIGHT™ tags enabled relative quantification between two samples.
Similar to the ethyl esterification reported for the released glycans , the method for the glycopeptide derivatization makes use of the carboxylic acid activator EDC and the catalyst HOBt. However, we found that the alcohols used for the esterification, while linkage-specific for the sialic acid, showed variable reactions on the peptide portion of the glycoconjugate. Trying different nucleophiles, including alcohols and amines and different conditions yielded a protocol employing dimethylamidation of the glycopeptides. The dimethylamidation showed selectivity for the sialic acid linkage (introducing a mass difference of 45.06 Da) as well as for the individual carboxylic acids on the peptide portion. The protocol proved highly repeatable for the subclass-specific glycosylation analysis of tryptic IgG glycopeptides. Finally, for two monoclonal antibodies with various sialic acid linkages, the profile of the glycopeptides after derivatization was found to be comparable to the released glycan profile.
Here, we present approaches for the linkage-specific sialic acid derivatization of both glycans and glycopeptides followed by mass spectrometric glycosylation analysis. The methods are fast, have excellent intra- and inter-day repeatability and make use of relatively inexpensive chemicals. We envision the use of these methods for the monitoring of protein glycosylation by MALDI-TOF-MS, both in the analysis of biopharmaceuticals and for the detection of glycomic disease biomarkers.
After released glycan derivatization we were able to study total N-glycomes of complex samples like human plasma, which can be used to compare samples from large patient cohorts to healthy controls in order to find specific biomarkers.
The derivatization method for glycopeptides has considerable potential for future site-, subclass- and sialic acid-linkage specific glycosylation profiling of therapeutic antibodies, as well as for biomarker discovery in clinical IgG samples derived from plasma.
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