The Minimum Information Required for a Glycomics Experiment (MIRAGE) Project: Improving the Standards for Reporting Mass-spectrometry-based Glycoanalytic Data

Kolarich, Daniel; Rapp, Erdmann; Struwe, Weston B.; Haslam, Stuart M.; Zaia, Joseph; McBride, Ryan; Agravat, Sanjay; Campbell, Matthew P.; Kato, Masaki; Ranzinger, René; Kettner, Carsten; York, William S.

doi:10.1074/mcp.o112.026492

Cited by 110 publications

(87 citation statements)

References 6 publications

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“…Relative quantification is done in relation to the sum of all identified glycoprotein forms using peak intensity values GalNAc residue rather than on the galactose. The fully sialylated core-2 glycan was also found sulfated, however, [17,18]. Chymotryptic peptides were separated on a Bpreparativeĉ olumn using a proportionally scaled up gradient as used for capillary LC-ESI-MS. For correct pooling, all fractions were inspected by LC-ESI-MS on the Micromass Ultima Q-TOF instrument.…”

Section: Analysis Of Released Glycansmentioning

confidence: 99%

Detailed characterization of the O-linked glycosylation of the neuropilin-1 c/MAM-domain

et al. 2015

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Neuropilins are involved in angiogenesis and neuronal development. The membrane proximal domain of neuropilin-1, called c or MAM domain based on its sequence conservation, has been implicated in neuropilin oligomerization required for its function. The c/MAM domain of human neuropilin-1 has been recombinantly expressed to allow for investigation of its propensity to engage in molecular interactions with other protein or carbohydrate components on a cell surface. We found that the c/MAM domain was heavily O-glycosylated with up to 24 monosaccharide units in the form of disialylated core 1 and core 2 O-glycans. Attachment sites were identified on the chymotryptic c/MAM peptide ETGATEKPTVIDSTIQSEFPTY by electron-transfer dissociation mass spectrometry (ETD-MS/MS). For highly glycosylated species consisting of carbohydrate to about 50 %, useful results could only be obtained upon partial desialylation. ETD-MS/MS revealed a hierarchical order of the initial O-GalNAc addition to the four different glycosylation sites. These findings enable future functional studies about the contribution of the described glycosylations in neuropilin-1 oligomerization and the binding to partner proteins as VEGF or galectin-1.As a spin-off result the sialidase from Clostridium perfringens turned out to discriminate between galactose- and N-acetylgalactosamine-linked sialic acid.

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Section: Analysis Of Released Glycansmentioning

confidence: 99%

Detailed characterization of the O-linked glycosylation of the neuropilin-1 c/MAM-domain

et al. 2015

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“…The released N-glycans were analyzed as described in detail in a MIRAGE (36) compliant manner in the supplementary material (29,37). Relative quantitation was performed using the Quant Analysis tool (Bruker), which determines the area under the curve obtained from the individual extracted ion chromatograms from multiple analyses (Supplementary Table S1).…”

Section: Glycoprofilingmentioning

confidence: 99%

An Anti-EGFR IgA That Displays Improved Pharmacokinetics and Myeloid Effector Cell Engagement In Vivo

et al. 2016

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Antibodies of IgA isotype effectively engage myeloid effector cells for cancer immunotherapy. Here, we describe preclinical studies with an Fc engineered IgA2m(1) antibody containing the variable regions of the EGFR antibody cetuximab. Compared with wild-type IgA2m(1), the engineered molecule lacked two Nglycosylation sites (N166 and N337), two free cysteines (C311 and C472), and contained a stabilized heavy and light chain linkage (P221R mutation). This novel molecule displayed improved production rates and biochemical properties compared with wild-type IgA. In vitro, Fab-and Fc-mediated effector functions, such as inhibition of ligand binding, receptor modulation, and engagement of myeloid effector cells for antibody-dependent cell-mediated cytotoxicity, were similar between wild-type and engineered IgA2. The engineered antibody displayed lower levels of terminal galactosylation leading to reduced asialoglycoproteinreceptor binding and to improved pharmacokinetic properties. In a long-term in vivo model against EGFR-positive cancer cells, improved serum half-life translated into higher efficacy of the engineered molecule, which required myeloid cells expressing human FcaRI for its full efficacy. However, Fab-mediated effector functions contributed to the in vivo efficacy because the novel IgA antibody demonstrated therapeutic activity also in nonFcaRI transgenic mice. Together, these results demonstrate that engineering of an IgA antibody can significantly improve its pharmacokinetics and its therapeutic efficacy to inhibit tumor growth in vivo. Cancer Res; 76(2); 403-17. Ó2015 AACR.

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“…Optimization of sample preprocessing procedures appears not to be sufficient and it may be necessary to apply uniform computational processing of the experimentally obtained data. In that context, the minimum information required for a glycomics experiment (MIRAGE) initiative represents a first important step forward towards standardized reporting of experimental conditions [20, 21]; http://www.beilstein-institut.de/en/projects/mirage). …”

Section: Discussionmentioning

confidence: 99%

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines

et al. 2015

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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 in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples. KeywordsHuman proteome organization (HUPO); Human disease glycomics/proteome initiative (HGPI); Glycoproteomics Ito et al.

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The Minimum Information Required for a Glycomics Experiment (MIRAGE) Project: Improving the Standards for Reporting Mass-spectrometry-based Glycoanalytic Data

Cited by 110 publications

References 6 publications

Detailed characterization of the O-linked glycosylation of the neuropilin-1 c/MAM-domain

Detailed characterization of the O-linked glycosylation of the neuropilin-1 c/MAM-domain

An Anti-EGFR IgA That Displays Improved Pharmacokinetics and Myeloid Effector Cell Engagement In Vivo

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines

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