Guinevere S.M. Kammeijer (1), David Falck (1), Bas C. Jansen (1), Paul J. Hensbergen (1), Manfred Wuhrer (1,2)
(1) Center for Proteomics and Metabolomics, Leiden University Medical Center (2) Division of BioAnalytical Chemistry, VU University Amsterdam
A powerful analytical platform for the analysis of biomolecules is capillary electrophoresis – electrospray ionization – mass spectrometry (CESI-MS). Implementation of an acetonitrile-enriched nebulizer gas on the CESI-MS system yielded a more robust platform as well as a gain in sensitivity compared to the conventional platform. The gain in sensitivity could aid in the analysis of samples which are only available in minute amounts. The conventional CESI-MS method was used for the analysis of the glycoprotein prostate specific antigen (PSA). We were able to identify 50 different N-glycans on a single N-glycosylation site (N69). The used method shows great potential for studying PSA in prostate cancer research to identify disease related alterations of glycosylation in an early stage and therefore could be a promising tool for diagnostic and prognostic evaluation.
A powerful analytical platform for the analysis of biomolecules is capillary electrophoresis – electrospray ionization – mass spectrometry (CESI-MS), the hyphenated form of CE and ESI shows high separation efficiency and sensitivity. To boost the sensitivity and robustness of the CESI interface, we have commenced an explorative study of combining a solvent-doped nebulizer gas with a sheathless ESI interface, which has shown a considerably improved ionization of glycopeptides in nanospray LC-ESI-MS . To this end, we used the glycoprotein immunoglobulin(Ig)G, which is well characterized and contains only one N-linked glycosylation site [2, 3]. The purified and enriched IgG glycopeptides were acquired by tryptic digest and cotton hydrophilic interaction liquid chromatography (HILIC). The sample was analysed with CESI-MS including and excluding the employment of a nebulizer gas which consisted of acetonitrile-enriched nitrogen gas.
Results showed that we could obtain a 9-fold increase in the area under the curve (AUC) of the 10 most abundant glycopeptides of IgG1 using the acetonitrile-enriched nebulizer gas with concomitant optimized MS-settings. The averaged relative standard deviation was reduced from 7.9% to 0.7%, indicating a more robust platform for analyses. Further analyses are in progress to investigate the reproducibility of the enhanced glycopeptide ionization.
Additionally, we have studied the glycoprotein prostate specific antigen (PSA) with the conventional CESI-MS platform. As an early detection method for prostate cancer (PCa) the concentration of PSA in the circulation is measured. However, this method lacks specificity for PCa, as increased PSA concentrations can also be found with benign hyperplasia. The lack of specificity indicates the necessity for a more specific test to prevent unnecessary biopsies. Previous studies have reported that PCa can be distinguished from benign hyperplasia by investigating the post translational modification of PSA, namely glycosylation [4-6]. To date, the used methods for determining the glycosylation of PSA suffer from interferences from other glycoproteins. This study addresses the mentioned shortcoming by using the powerful analytical CESI-MS platform for the analysis of these biomolecules.
Analysis showed that the glycopeptides were mainly separated based on the glycan composition, especially the degree of sialylation. Sialic acids are often found at the terminus of the glycan chain, serving as a binding site for human lectins, toxins and pathogens. Literature suggests that the linkages of sialic acid can be an important marker of disease progression . We observed that with our current method we are able to separate the isomers of sialylated glycopeptide species. This separation could be of importance as α2,3-linked sialic acids seems to be a hallmark of malignant PCa. This can be explained by the fact that α2,3-linked sialic acids are needed for the formation of sialyl-LewisX structures which are implicated in cellular motility . The observed separation was confirmed by the analysis of IgG Fc glycopeptides from recombinant IgG (α2,3-linked sialic acids) and human plasma IgG (α2,6-linked sialic acids).
In summary, implementation of the acetonitrile-enriched nebulizer gas on the CESI-MS platform and optimized settings yielded a more robust platform as well as a gain in sensitivity compared to the conventional platform. The gain in sensitivity could aid in the analysis of samples which are only available in minute amounts. With the conventional CESI-MS method we were able to distinguish between 50 different N-glycans from a single N-glycosylation site (N69) of PSA. The used method shows great potential for studying PSA in PCa research to identify disease related alterations of glycosylation. The measurement of altered glycosylation could be a promising tool for diagnostic and prognostic evaluation.
. Marx, K., A. Kiehne, and M. Meyer, Application Note #LCMS-93; amaZon speed ETD: Exploring glycopeptides in protein mixtures using Fragment Triggered ETD and CaptiveSpray nanoBooster. 2014, Bruker Daltonik GmbH.
. Heemskerk, A.A.M., et al., Coupling porous sheathless interface MS with transient-ITP in neutral capillaries for improved sensitivity in glycopeptide analysis. ELECTROPHORESIS, 2013. 34(3): p. 383-387.
. Jayo, R.G., et al., Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface. Analytical Chemistry, 2014. 86(13): p. 6479-6486.
. Janković, M.M. and M.M. Kosanović, Glycosylation of urinary prostate-specific antigen in benign hyperplasia and cancer: assessment by lectin-binding patterns. Clinical Biochemistry, 2005. 38(1): p. 58-65.
. Peracaula, R., et al., Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins. Glycobiology, 2003. 13(6): p. 457-470.
. Saldova, R., et al., Core fucosylation and α2-3 sialylation in serum N-glycome is significantly increased in prostate cancer comparing to benign prostate hyperplasia. Glycobiology, 2011. 21(2): p. 195-205.
. Alley, W.R. and M.V. Novotny, Glycomic Analysis of Sialic Acid Linkages in Glycans Derived from Blood Serum Glycoproteins. Journal of Proteome Research, 2010. 9(6): p. 3062-3072.
. Jørgensen, T., et al., Up-Regulation of the Oligosaccharide Sialyl LewisX: A New Prognostic Parameter in Metastatic Prostate Cancer. Cancer Research, 1995. 55(9): p. 1817-1819.