Workflow for combined proteomics and glycomics profiling from histological tissues (original) (raw)
Related papers
Glycobiology, 2007
Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/ Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.
Molecular & Cellular Proteomics, 2013
Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either Nglycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of Nglycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed deN -glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including deN -glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their non-sugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, protein quantification based exclusively upon deglycosylated peptides may lead to inaccurate quantification.
Glycomics meets genomics, epigenomics and other high throughput omics for system biology studies
2013
Majority of eukaryotic proteins are glycosylated and their glycan moieties have numerous important structural, functional and regulatory roles. Because of structural complexity of glycans and technological limitations glycomics, and particularly glycoproteomics was not able to follow rapid progress in genomics and proteomics over last 30 years. However, the field of glycan has been progressing rapidly and first large-scale studies of the glycome have been completed recently. These studies have revealed significant differences in glycome composition between individuals, which may contribute to the human phenotypic variability. The current state-of-the-art in high-throughput glycomics and its integration with genomics, epigenomics and lipidomics is reviewed in this article.
Glycoconjugate journal, 2015
The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared ...
Towards automation of glycomic profiling of complex biological materials
Glycoconjugate Journal, 2018
Glycosylation is considered one of the most complex and structurally diverse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious sample preparation steps. Therefore, in this study, we aim to improve sample preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein/protein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of samples proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.
Molecular & Cellular Proteomics, 2009
Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and highthroughput manner. Thus, the established system should provide a powerful pipeline in support of ongoing efforts in glyco-biomarker discovery. Molecular & Cellular Proteomics 8:99-108, 2009. Glycan synthesis in individual cells is regulated by harmonized expression of more than a hundred glycosyltransferases. Importantly, detectable dynamics occurring on each cell surface during diverse biological events, e.g. differentiation, proliferation, and signal induction, indicate drastic changes in the glyco-machinery (1). During the last few decades, there have been enormous advances in the findings of glycosylation alterations related to oncogenesis. Cell surface sialylation and 1-6 branching of N-linked glycans are strongly correlated with metastatic potential of cancer cells (2, 3). Metastatic ability is also reflected by dramatic alteration of core structures of O-glycans (4). These observations suggest that novel tumor-specific glycoproteins accompanying substantial structural changes in glycan moieties will become reliable biomarkers with higher specificity than those established previously, e.g. CA19-9 (5), carcino-embryonic antigen family (6), ␣-fetoprotein (AFP) 1 (7), and prostate-specific antigen (PSA) (8). Recent advances in technologies such as mass spectrometry (MS), microarray, and laser microdissection have strongly advanced the proteomics-based biomarker discovery phase targeting both cultured cells and tissue specimens (9-14). This has resulted in the emergence of an extensive range of biomarker "candidates", many of which are glycoproteins. However, these candidate molecules need to be subsequently subjected to a verification step prior to a much large scale validation phase (e.g. treating with Ͼ1,000 incidences). Antibody microarray has taken the place of ELISA as a more versatile and high-throughput technique, and has enabled multiplexed quantitative analysis for over a hundred proteins with sufficient sensitivity (15). As an application of MS technology, multiple-reaction monitoring-MS has also been developed (16-19). However, taking into consideration the critical glycosylation changes occurring on diverse glycoproteins, differential analysis of respective glycans is necessary in parallel with quantitative protein analysis. In this context, Chen et al. (20) have recently developed a lectin-overlay antibody microarray with the intention of discovering glycoprotein biomarkers. However, the proposed strategy requires repeated
International Journal of Mass Spectrometry, 2012
The biological functions of glycoconjugate glycans arise in the context of structural heterogeneity resulting from non-template driven biosynthetic reactions. Such heterogeneity is particularly apparent for the glycosaminoglycan (GAG) classes, of which heparan sulfate (HS) is of particular interest for its properties in binding to many classes of growth factors and growth factor receptors. The structures of HS chains vary according to spatial and temporal factors in biological systems as a mechanism where by the functions of the relatively limited number of associated proteoglycan core proteins is elaborated. Thus, there is a strong driver for the development of methods to discover functionally relevant structures in HS preparations for different sources. In the present work, a set of targeted tandem mass spectra were acquired in automated mode on HS oligosaccharides deriving from two different tissue sources. Statistical methods were used to determine the precursor and product ions, the abundances of which differentiate between the tissue sources. The results demonstrate considerable potential for using this approach to constrain the number of positional glycoform isomers present in different biological preparations toward the end of discovery of functionally relevant structures.
18 O Labeling for a Quantitative Proteomic Analysis of Glycoproteins in Hepatocellular Car
Clinical Proteomics, 2008
Introduction Quantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectromet-ric analyses increases the likelihood of identifying cancerspecific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers. Materials and Methods In this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent 18 O/ 16 O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to 18 O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results. Conclusion This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.
Site-Specific Glycan-Peptide Analysis for Determination of N -Glycoproteome Heterogeneity
Journal of Proteome Research, 2013
A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography−tandem mass spectrometry (PGC-LC−MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove Nglycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the Nglycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.