Application Note 37
Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT™): A Novel N-Linked Glycan Relative Quantification Strategy
David C. Muddiman and Amber C. Cook
W.M. Keck Fourier Transform Mass Spectrometry Laboratory, Department of Chemistry
North Carolina State University, Raleigh, NC 27695 USA
Bioanalytical characterization and quantification of protein post-translation modifications (PTMs) is fundamental to elucidating the functions and activities of PTMs in mediating intra- and extra-cellular interactions. More importantly, how and to what degree do post-translational modifications participate in mediating human health as well as change in response to illness or disease? In a typical discovery-based, bottom-up proteomics experiment (i.e., whole proteome is digested with a proteolytic enzyme), one can often detect the presence of PTMs, including phosphorylation, glycosylation, acetylation and ubiquitination.
Biomarker-based mass spectrometry has evolved exponentially over the past 20 years, facilitating in-depth analysis of biological samples. Moreover, comparative genomics, transcriptomics, proteomics, glycomics and metabolomics have increased awareness of the integrated complexity found in all biological systems. However, to enable the detection of PTMs at a depth sufficient to understand the underlying biology and function or dysfunction of a specific protein or in a proteome-wide measurement (global proteomics), a specific PTM of interest must be approached in a targeted fashion. The challenge of distinguishing healthy and diseased populations is embraced through collaborative efforts to quantify biological change at the molecular level.
N-Linked glycosylation is known to be involved in the regulatory affairs of a proteome, weakening the ability to distinguish abnormal and normal, in terms of protein structure and function. N-Linked glycosylation is a very common PTM, and it has been estimated that 60-90% of all mammalian proteins are glycosylated while membrane and secreted proteins are almost always glycosylated.1 Glycosylation is a key PTM, mediating both intra-cellular and extra-cellular communication; principally, at the protein level: regulation of folding, intra-cellular trafficking, secretion and extra-cellular recognition (e.g., cell adhesion and immune response.2,3 These key physiological roles of glycosylation have prompted active research in what is collectively known as the field of glycomics.4-6
Numerous strategies may be used to study glycosylation including top-down approaches which invoke lectin affinity enrichment of the glyco-proteome or a bottom-up approach, digesting the glycoproteome into glycopeptides that are subsequently enriched by lectin chromatography; resultant peptides are then subjected to LC-MS analysis. Alternatively whole glycans may be cleaved and isolated at the protein and/or peptide levels, thus generating a global view of the entire glycome from which the glycans are derived.7
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