Label-free quantitation: A new glycoproteomics approach

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Glycopeptide-based analysis is used to inform researchers about the glycans on one or more proteins. The method's key attractive feature is its ability to link glycosylation information to exact locations (glycosylation sites) on proteins. Numerous applications for glycopeptide analysis are known, and several examples are described herein. The techniques used to characterize glycopeptides are still emerging, and recently, research focused on facilitating aspects of glycopeptide analysis has advanced significantly in the areas of sample preparation, MS fragmentation, and automation of data analysis. These recent developments, described herein, provide the foundation for the growth of glycopeptide analysis as a blossoming field. Molecular & Cellular

Section: Emerging Methods Supporting Glycopeptide Analysismentioning

confidence: 99%

Glycopeptide Analysis, Recent Developments and Applications

Desaire

2013

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“…In a different approach, the SILAC technique allows incorporation of stable isotope-labeled amino acids into proteins during cell culturing process (196), and was applied to investigate the difference in cell surface N-glycoproteins among different cell types (64). A label-free approach has also been used for glycoproteomics profiling, including a method developed to profile intact glycopeptides in a complex sample (208) and a study that compares the plasma glycoproteome between psoriasis patients and healthy controls (209).…”

mentioning

confidence: 99%

Mass Spectrometry Based Glycoproteomics—From a Proteomics Perspective

Pan

Chen

Aebersold

et al. 2011

249

239

Glycosylation is one of the most important and common forms of protein post-translational modification that is involved in many physiological functions and biological pathways. Altered glycosylation has been associated with a variety of diseases, including cancer, inflammatory and degenerative diseases. Glycoproteins are becoming important targets for the development of biomarkers for disease diagnosis, prognosis, and therapeutic response to drugs. The emerging technology of glycoproteomics, which focuses on glycoproteome analysis, is increasingly becoming an important tool for biomarker discovery. An in-depth, comprehensive identification of aberrant glycoproteins, and further, quantitative detection of specific glycosylation abnormalities in a complex environment require a concerted approach drawing from a variety of techniques. This report provides an overview of the recent advances in mass spectrometry based glycoproteomic methods and technology, in the context of biomarker discovery and clinical application. Molecular & Cellular

“…For alkylated IgA1 HR preparations, cysteine iodoacetamide modification was selected. The relative abundance of each glycopeptide was calculated by use of a label-free quantification method described by Rebecchi et al (37). After assigning all glycopeptide peaks in the spectrum, the ion chromatogram for each glycopeptide ion was individually extracted for the specific glycopeptide ion species Ϯ0.83 m/z.…”

mentioning

confidence: 99%

Clustered O-Glycans of IgA1

Takahashi

Wall

Suzuki

et al. 2010

Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1-5). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6). A subset of these glycoproteins has clustered sites of O-glycosylation with serine-and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7). Immunoglobulin A1 (IgA1) 1 contains both O-and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schö nlein purpura nephritis (1, 8). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9 -11). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12).An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques.