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Magnetic carbon nanocomposites as a MALDI co-matrix enhancing MS-based glycomics.

ABSTRACT: More than 50% of all known proteins are glycosylated, which is critical for many biological processes such as protein folding and signal transduction. Glycosylation has proven to be associated with different mammalian diseases such as breast and liver cancers. Therefore, characterization of glycans is highly important to facilitate a better understanding of the development and progression of many human diseases. Although matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) offers several advantages such as ease of operation and short analysis times, however, due to the complexity of glycan structures and their low ionization efficiency, there are still challenges that need to be addressed to achieve sensitive glycan analysis. Here, magnetic carbon nanocomposites (CNPs@Fe3O4 NCs) were used as a new MALDI matrix or co-matrix for the analysis of glycans derived from different model glycoproteins and human blood serum samples. The addition of CNPs@Fe3O4 NCs to the matrix significantly enhanced glycan signal intensity by several orders of magnitude, and effectively controlled/reduced/eliminated in-source decay (ISD) fragmentation. The latter was attained by modulating CNPs@Fe3O4 NCs concentrations and allowed the simultaneous study of intact and fragmented glycans, and pseudo-MS3 analysis. Moreover, CNPs@Fe3O4 NCs was also effectively employed to desalt samples directly on MALDI plate, thus enabling direct MALDI-MS analysis of unpurified permethylated glycans derived from both model glycoproteins and biological samples. On-plate desalting enhanced sensitivity by reducing sample loss. Graphical abstract ?.

SUBMITTER: Banazadeh A

PROVIDER: S-EPMC6375713 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Magnetic carbon nanocomposites as a MALDI co-matrix enhancing MS-based glycomics.

Banazadeh Alireza A Williamson Seth S Zabet Masoud M Hussien Ahmed A Mechref Yehia Y

Analytical and bioanalytical chemistry 20180908 28

More than 50% of all known proteins are glycosylated, which is critical for many biological processes such as protein folding and signal transduction. Glycosylation has proven to be associated with different mammalian diseases such as breast and liver cancers. Therefore, characterization of glycans is highly important to facilitate a better understanding of the development and progression of many human diseases. Although matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) of ...[more]

PMID: 30196422

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Project description:Highly specialized cells are fundamental for proper functioning of complex organs. Variations in cell-type specific gene expression and protein composition have been linked to a variety of diseases. Although single cell technologies have emerged as valuable tools to address this cellular heterogeneity, a majority of these workflows lack sufficient in situ resolution for functional classification of cells and are associated with extremely long analysis time, especially when it comes to in situ proteomics. In addition, lack of understanding of single cell dynamics within their native environment limits our ability to explore the altered physiology in disease development. This limitation is particularly relevant in the mammalian brain, where different cell types perform unique functions and exhibit varying sensitivities to insults. The hippocampus, a brain region crucial for learning and memory, is of particular interest due to its obvious involvement in various neurological disorders. Here, we present a combination of experimental and data integration approaches for investigation of cellular heterogeneity and functional disposition within the mouse brain hippocampus using MALDI Imaging mass spectrometry (MALDI-IMS) and shotgun proteomics (LC-MS/MS) coupled with laser-capture microdissection (LCM) along with spatial transcriptomics. Within the dentate gyrus granule cells we identified two proteomically distinct cellular subpopulations that are characterized by a substantial number of discriminative proteins. These cellular clusters contribute to the overall functionality of the dentate gyrus by regulating redox homeostasis, mitochondrial organization, RNA processing, and microtubule organization. Importantly, most of the identified proteins matched their transcripts, verifying the in situ protein identification and supporting their functional analyses. By combining high-throughput spatial proteomics with transcriptomics, our approach enables reliable near-single-cell scale identification of proteins and profiling of inter-cellular heterogeneity within similar cell-types in tissues. This methodology has the potential to be applied to different biological conditions and tissues, providing a deeper understanding of cellular subpopulations in situ.

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.

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Magnetic carbon nanocomposites as a MALDI co-matrix enhancing MS-based glycomics.

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Magnetic carbon nanocomposites as a MALDI co-matrix enhancing MS-based glycomics.

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