The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative to standardize the reporting of glycoanalytical methods and to assess their reproducibility. To date, the... Show moreThe Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative to standardize the reporting of glycoanalytical methods and to assess their reproducibility. To date, the MIRAGE Commission has published several reporting guidelines that describe what information should be provided for sample preparation methods, mass spectrometry methods, liquid chromatography analysis, exoglycosidase digestions, glycan microarray methods, and nuclear magnetic resonance methods. Here, we present the first version of reporting guidelines for glyco(proteo)mics analysis by capillary electrophoresis (CE) for standardized and high-quality reporting of experimental conditions in the scientific literature. The guidelines cover all aspects of a glyco(proteo)mics CE experiment including sample preparation, CE operation mode (CZE, CGE, CEC, MEKC, cIEF, cITP), instrument configuration, capillary separation conditions, detection, data analysis, and experimental descriptors. Show less
Iacomelli, I.; Barberio, G.; Pucci, P.; Monaco, V.; Maffei, M.; Mogni, M.; ... ; Ivaldi, G. 2021
Objectives: Artifactually altered glycated hemoglobin (HbA(1c)) concentrations are frequently linked to hemoglobin (Hb) variants. Their expression and detection require in-depth analysis.Methods:... Show moreObjectives: Artifactually altered glycated hemoglobin (HbA(1c)) concentrations are frequently linked to hemoglobin (Hb) variants. Their expression and detection require in-depth analysis.Methods: Cation exchange high performance liquid chromatography (HPLC) (Bio-Rad Variant (TM) II; Trinity Biotech Premier Hb9210 Resolution), capillary electrophoresis (CE) (Sebia Capillarys 2 Flex Piercing) and mass spectrometry (MS) (Waters) were used for variant detection; Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) were used for DNA analysis; HbA(1c) was measured with cation exchange HPLC (Bio-Rad Variant (TM) II; Arkray Adams HA-8180V; Tosoh HLC-723 G7), CE (Sebia Capillarys 2 Flex Piercing), boronate affinity HPLC (Trinity Biotech Hb9210 Premier), immunoassay (Cobas c501 Tina-quant HbA(1c) Gen. 3; Nihon Kohden CHM-4100 Celltac chemi HbA(1c) HA-411V) and enzymatic assay (Abbott Architect c 8000 HbA(1c)).Results: Hb Yamagata [beta 132(H10)Lys -> Asn; (HBB: c.399A>T)] was identified in the proband by MS after the observation of an abnormal peak in HPLC and CE. A mosaic expression of this variant was detected by NGS (mutant: 8%; wild type: 92%), after negative results in Sanger sequencing. Hb Yamagata interfered with HbA(1c) measurements by cation exchange HPLC and CE whereas immuno and enzymatic assay values showed good agreement with boronate affinity HPLC measurement.Conclusions: A mosaicism of Hb Yamagata was found in a patient with altered HbA(1c) values. This rare gene variant was detected only by advanced technologies as MS and NGS. The variant interfered with common HbA(1c) determination methods. Show less
Current approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they... Show moreCurrent approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they also result in a loss of valuable information regarding certain subclasses, allotypes and co-occuring post-translational modifications (PTMs). Unfortunately, the high variability of polyclonal IgGs makes their intact mass spectrometry (MS) analysis extremely challenging. We propose here a middle-up strategy for the analysis of the intact fragment crystallizable (Fc) region of human plasma IgGs, with the aim of acquiring integrated information of theN-glycosylation and other PTMs of subclasses and allotypes. Human plasma IgG was isolated using Fc-specific beads followed by an on-bead C(H)2 domain digestion with the enzyme IdeS. The obtained mixture of Fc subunits was analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) hyphenated with MS. CE-MS provided separation of different IgG-subclasses and allotypes, while HILIC-MS allowed resolution of the different glycoforms and their oxidized variants. The orthogonality of these techniques was key to reliably assign Fc allotypes. Five individual donors were analyzed using this approach. Heterozygosis was observed in all the analyzed donors resulting in a total of 12 allotypes identified. The assignments were further confirmed using recombinant monoclonal IgG allotypes as standards. While the glycosylation patterns were similar within allotypes of the same subclass, clear differences were observed between IgG subclasses and donors, highlighting the relevance of the proposed approach. In a single analysis, glycosylation levels specific for each allotype, relative abundances of subclasses and information on co-occurring modifications are obtained. This middle-up method represents an important step toward a comprehensive analysis of immunoglobulin G-Fc variants. Show less
Kohler, I.; Verhoeven, A.; Derks, R.J.E.; Giera, M. 2016
Metabolomics-based strategies have become an integral part of modern clinical research, allowing for a better understanding of pathophysiological conditions and disease mechanisms, as well as... Show moreMetabolomics-based strategies have become an integral part of modern clinical research, allowing for a better understanding of pathophysiological conditions and disease mechanisms, as well as providing innovative tools for more adequate diagnostic and prognosis approaches. Metabolomics is considered an essential tool in precision medicine, which aims for personalized prevention and tailor-made treatments. Nevertheless, multiple pitfalls may be encountered in clinical metabolomics during the entire workflow, hampering the quality of the data and, thus, the biological interpretation. This review describes the challenges underlying metabolomics-based experiments, discussing step by step the potential pitfalls of the analytical process, including study design, sample collection, storage, as well as preparation, chromatographic and electrophoretic separation, detection and data analysis. Moreover, it offers practical solutions and strategies to tackle these challenges, ensuring the generation of high-quality data. Show less
![Hemoglobin Yamagata [beta 132(H10)Lys -> Asn; (HBB: c.399A > T)]: a mosaic to be put together](/sites/all/modules/custom/islandora_solr_search/images/defaultimg.png?solr_nav%5Bid%5D=5aa845e9983a65f3e596&solr_nav%5Bpage%5D=0&solr_nav%5Boffset%5D=1)
