Recent advances in the MS analysis of glycoproteins: Theoretical considerations.
ABSTRACT Protein glycosylation is involved in a broad range of biological processes that regulate protein function and control cell fate. As aberrant glycosylation has been found to be implicated in numerous diseases, the study and large-scale characterization of protein glycosylation is of great interest not only to the biological and biomedical research community, but also to the pharmaceutical and biotechnology industry. Due to the complex chemical structure and differing chemical properties of the protein/peptide and glycan moieties, the analysis and structural characterization of glycoproteins has been proven to be a difficult task. Large-scale endeavors have been further limited by the dynamic outcome of the glycosylation process itself, and, occasionally, by the low abundance of glycoproteins in biological samples. Recent advances in MS instrumentation and progress in miniaturized technologies for sample handling, enrichment and separation, have resulted in robust and compelling analysis strategies that effectively address the challenges of the glycoproteome. This review summarizes the key steps that are involved in the development of efficient glycoproteomic analysis methods, and the latest innovations that led to successful strategies for the characterization of glycoproteins and their corresponding glycans. As a follow-up to this work, we review innovative capillary and microfluidic-MS workflows for the identification, sequencing and characterization of glycoconjugates.
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ABSTRACT: Glycobioinformatics is a rapidly developing field providing a vital support for mass spectrometry based glycoproteomics research. Recent advances in mass spectrometry greatly increased technological capabilities for high throughput glycopeptide analysis. However, interpreting mass spectrometry output, in terms of identifying glycan structures, attachment sites and glycosylation linkages still presents multiple challenges. Here, we discuss current strategies used in mass spectrometry based glycoproteomics and bioinformatics tools available for mass spectrometry based glycopeptide and glycan analysis. We also provide a brief overview of recent efforts in glycobioinformatics such as the new initiative UniCarbKB directed toward developing more comprehensive and unified glycobioinformatics platforms. With regards to glycobioinformatics tools and applications, we do not express our personal preferences or biases, but rather focus on providing a concise description of main features and functionalities of each application with the goal of assisting readers in making their own choices and identifying and locating glycobioinformatics tools most suitable for achieving their experimental objectives.Proteomics 01/2013; 13(2). DOI:10.1002/pmic.201200149 · 3.97 Impact Factor
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ABSTRACT: Tandem MS is considered as a mass spectrum of a mass spectrum raised to the power n, where n is the number of MS/MS spectra, obtained per molecular ion. When a peptide is analyzed by mass spectrometry, the obtained MS spectrum provides the peptide mass. When tandem MS (or MS/MS) is performed, such peptide is fragmented into daughter ions, which provide information regarding the amino acid sequence of the peptide. Due to this particularity, peptide tandem MS can be used in several applications involving protein characterization and identification, such as the study of proteomes (Franco et al., 2011a,b) and differential proteomics (Puerto et al., 2011) of different organs, tissues, cells or biological fluids. In our laboratory we have also been using this approach for the characterization of proteins with specific function in adhesives (Santos et al., 2009), the identification of protein adducts as potential biomarkers of toxicity (Antunes et al., 2010), and protein glycation (Gomes et al., 2008), it has also been useful in the identification of peptides with immunomodulation properties (Koči et al., 2010). Two types of information obtained by mass spectrometry experiments are used for the study of proteins and peptides. They can be identified or characterized based on their peptide map and primary structure. The advantage of using tandem MS, is that it provides further data, and hence, confirm the assigned peptide identification, thus reducing the chance of obtaining wrongly assigned peptide/protein identifications. Additionally, the information obtained allows localizing of post-translational or chemical modifications at the amino acid residue level.1 edited by Jeevan Prasain, 02/2012; InTech Publishers., ISBN: 978-953-51-0141-3
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ABSTRACT: Despite the importance of protein N-glycosylation in a series of biological processes, in-depth characterization of the protein glycosylation is still a challenge due to the high complexity of biological samples and the lacking of highly-sensitive detection technologies. We developed a monolithic capillary column based glycoproteomic reactor enabling high-sensitive mapping of N-glycosylation sites from minute amount of sample. Unlike the conventional proteomic reactors with only strong-cation exchange (SCX) or hydrophilic interaction chromatography (HILIC) column, this novel glycoproteomic reactor was composed of an 8 cm-long C12 hydrophobic monolithic capillary column for protein digestion and a 6 cm-long organic-silica hybrid hydrophilic monolithic capillary column for glycopeptides enrichment and deglycosylation, which could complete whole sample preparation including protein purification/desalting, tryptic digestion, enrichment and deglycosylation of glycopeptides within about 3 h. The developed reactor exhibited high detection sensitivity in mapping of N-glycosylation sites by detection limit of HRP as low as 2.5 fmol. This reactor also demonstrated the ability in complex sample analysis, and totally 486 unique N-glycosylation sites were reliably mapped in three replicate analyses of protein sample extracted from ~104 HeLa cells.Analytical Chemistry 02/2013; 85(5). DOI:10.1021/ac400315n · 5.83 Impact Factor