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Sensitive and Structure-Informative N-Glycosylation Analysis by MALDI-MS; Ionization, Fragmentation, and Derivatization.

ABSTRACT: Mass spectrometry (MS) has become an indispensable tool for analyzing post translational modifications of proteins, including N-glycosylated molecules. Because most glycosylation sites carry a multitude of glycans, referred to as "glycoforms," the purpose of an N-glycosylation analysis is glycoform profiling and glycosylation site mapping. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has unique characteristics that are suited for the sensitive analysis of N-glycosylated products. However, the analysis is often hampered by the inherent physico-chemical properties of N-glycans. Glycans are highly hydrophilic in nature, and therefore tend to show low ion yields in both positive- and negative-ion modes. The labile nature and complicated branched structures involving various linkage isomers make structural characterization difficult. This review focuses on MALDI-MS-based approaches for enhancing analytical performance in N-glycosylation research. In particular, the following three topics are emphasized: (1) Labeling for enhancing the ion yields of glycans and glycopeptides, (2) Negative-ion fragmentation for less ambiguous elucidation of the branched structure of N-glycans, (3) Derivatization for the stabilization and linkage isomer discrimination of sialic acid residues.

SUBMITTER: Nishikaze T

PROVIDER: S-EPMC5543250 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Project description:Sensitive glycomics analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is of great importance but significantly hampered by their low ionization efficiency and labile sialic acid moieties. Chemical derivatization offers a viable way to improve both the ionization efficiency and analytical sensitivity of the glycans in MS analysis by altering their hydrophobicity or charge property. Here we employed Girard's reagent T (GT) for on-target derivatization (GTOD) of reducing glycan under mild acid condition to form stable hydrazones at room temperature, allowing rapid and sensitive identification of neutral and sialylated glycans in positive-ion mode as only permanently positive charged molecular ions without multiple ion adducts by MALDI-TOF-MS. The MS signal intensities of lactose, sialylated N-glycans derived from bovine fetuin and neutral N-glycans derived from RNaseB and ovalbumin were boosted by 7.44, 9.13, 12.96 and 13.47 folds on average (n?=?3), respectively. More importantly, after GTOD strategy, unwanted desialylation of sialylated glycans during MS was suppressed. The detection limit of the assay is desirable since the nanogram of N-glycans derived from 0.16??g ovalbumin could be detected. The assay demonstrated good stability (RSD?2.95%, within 10 days), reliable reproducibility (RSD?=?2.96%, n?=?7) and a desirable linear dynamic range from 78?nmol/mL to 10??mol/mL. The strategy has been successfully applied to MS analysis of reducing glycans from human milks, neutral and sialylated O-, N-glycans from glycoproteins, and reducing glycans derived from glycosphingolipids, presenting neater [M]+ signals which allow detection of more low-abundance glycans and assignation of Neu5Ac vs. Neu5Gc or fucose vs. hexose in glycans due to the absence of the ambiguous interpretation from multiple peaks (ion adducts [M+Na]+ and [M+K]+). Moreover, the GTOD assay prevents desialylation during MALDI-TOF-MS profiling and enables distinct linkage-specific characterization of terminal sialic acids of N-glycans derived from human serum protein when combines with an esterification.

Project description:The ability to characterize chemical heterogeneity in biological structures is essential to understanding cellular-level function in both healthy and diseased states, but these variations remain difficult to assess using a single analytical technique. While mass spectrometry (MS) provides sufficient sensitivity to measure many analytes from volume-limited samples, each type of mass spectrometric analysis uncovers only a portion of the complete chemical profile of a single cell. Matrix-assisted laser desorption/ionization (MALDI) MS and capillary electrophoresis electrospray ionization (CE-ESI)-MS are complementary analytical platforms frequently utilized for single-cell analysis. Optically guided MALDI MS provides a high-throughput assessment of lipid and peptide content for large populations of cells, but is typically nonquantitative and fails to detect many low-mass metabolites because of MALDI matrix interferences. CE-ESI-MS allows quantitative measurements of cellular metabolites and increased analyte coverage, but has lower throughput because the electrophoretic separation is relatively slow. In this work, the figures of merit for each technique are combined via an off-line method that interfaces the two MS systems with a custom liquid microjunction surface sampling probe. The probe is mounted on an xyz translational stage, providing 90.6 ± 0.6% analyte removal efficiency with a spatial targeting accuracy of 42.8 ± 2.3 μm. The analyte extraction footprint is an elliptical area with a major diameter of 422 ± 21 μm and minor diameter of 335 ± 27 μm. To validate the approach, single rat pancreatic islet cells were rapidly analyzed with optically guided MALDI MS to classify each cell into established cell types by their peptide content. After MALDI MS analysis, a majority of the analyte remains for follow-up measurements to extend the overall chemical coverage. Optically guided MALDI MS was used to identify individual pancreatic islet α and β cells, which were then targeted for liquid microjunction extraction. Extracts from single α and β cells were analyzed with CE-ESI-MS to obtain qualitative information on metabolites, including amino acids. Matching the molecular masses and relative migration times of the extracted analytes and related standards allowed identification of several amino acids. Interestingly, dopamine was consistently detected in both cell types. The results demonstrate the successful interface of optical microscopy-guided MALDI MS and CE-ESI-MS for sequential chemical profiling of individual, mammalian cells.

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Sensitive and Structure-Informative N-Glycosylation Analysis by MALDI-MS; Ionization, Fragmentation, and Derivatization.

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