On the basis of glycan derivatization principles described in literature, we developed a method for the linkage specific derivatization of glycans.1 This method has various advantageous features:
– Sialic acids are stabilized allowing their robust detection by MALDI-TOF-MS
– Sialic acid linkages are differentiated – which is very important due to their different biological functions.
– The technique works is 96-well plate format with MALDI-TOF-MS detection, allowing the facile analysis of large glycoprotein sample sets.
– We successfully robotized the method, resulting in an increase in throughput and largely eliminating inter-operator variation.2
– The technique works on impure samples. This means that, after enzymatic glycan release from glycoproteins, glycans can be derivatized right away without the need of purification before derivatization.
We adapted the derivatization to the analysis of IgG Fc glycopeptides, allowing the site-specific analysis of IgG Fc glycosylation by MALDI-TOF-MS with detection of sialic acids in a linkage-specific manner.3 This technique has been successfully applied to analyze IgG Fc glycosylation in children.4
At CPM, we use complementary mass spectrometry strategies to perform in-depth analysis and characterization of proteins from both complex biological specimens as well as pharmacological formulations. Intact protein analysis by mass spectrometry provides information about the amino acidic sequence and post-translational modifications in a single experiment. This so-called top-down analysis requires ultrahigh resolution mass analyzers and has been applied in our laboratories to localize chemical modifications (e.g. fluorescent labels), identify new proteoforms and glycosylation, and confirm cysteine connectivities (i.e. disulfide bonds). Top-down MS is suitable for the analysis of proteins from any biological source under the condition that proteins from complex mixtures must be purified (or separated) prior to analysis. In our lab, we have analyzed intact proteins from human serum,5 bacterial extracts,6 protein standards7 and pharmaceuticals.8
- Reiding, K. R., Blank, D., Kuijper, D. M., Deelder, A. M. & Wuhrer, M. High-Throughput Profiling of Protein N-Glycosylation by MALDI-TOF-MS Employing Linkage-Specific Sialic Acid Esterification. Anal. Chem. 86, 5784-5793 (2014).
- Bladergroen, M. R. et al. Automation of high-throughput mass spectrometry-based plasma N-glycome analysis with linkage-specific sialic acid esterification. J. Proteome Res. 14, 4080-4086 (2015).
- de Haan, N. et al. Linkage-specific sialic acid derivatization for MALDI-TOF-MS profiling of IgG glycopeptides. Anal. Chem. 87, 8284-8291 (2015).
- de Haan, N., Reiding, K. R., Driessen, G., van der Burg, M. & Wuhrer, M. Changes in healthy human IgG Fc-glycosylation after birth and during early childhood. J. Proteome Res. 15, 1853-1861 (2016).
- Nicolardi, S., van der Burgt, Y. E., Dragan, I., Hensbergen, P. J. & Deelder, A. M. Identification of new apolipoprotein-CIII glycoforms with ultrahigh resolution MALDI-FTICR mass spectrometry of human sera. J. Proteome Res. 12, 2260-2268 (2013).
- Fleurbaaij, F. et al. Typing Pseudomonas aeruginosa Isolates with Ultrahigh Resolution MALDI-FTICR Mass Spectrometry. Anal. Chem. 88, 5996-6003 (2016).
- Nicolardi, S., Switzar, L., Deelder, A. M., Palmblad, M. & van der Burgt, Y. E. Top-down MALDI-in-source decay-FTICR mass spectrometry of isotopically resolved proteins. Anal. Chem. 87, 3429-3437 (2015).
- Nicolardi, S., Deelder, A. M., Palmblad, M. & van der Burgt, Y. E. Structural analysis of an intact monoclonal antibody by online electrochemical reduction of disulfide bonds and Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 86, 5376-5382 (2014).
- Reiding, K. R., Lonardi, E., Hipgrave Ederveen, A.L., Wuhrer, M. Ethyl Esterification for MALDI-MS Analysis of Protein Glycosylation. Methods Mol. Biol. 1394,151-162 (2016)