| 1. |
- Balonova, Lucie, et al.
(författare)
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Characterization of protein glycosylation in Francisella tularensis subsp holarctica
- 2012
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 11:7
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Tidskriftsartikel (refereegranskat)abstract
- FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.015016, 1-12, 2012.
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| 2. |
- Gizaw, Solomon T., et al.
(författare)
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Highly Sensitive O-Glycan Profiling for Human Serum Proteins Reveals Gender-Dependent Changes in Colorectal Cancer Patients
- 2019
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Ingår i: Analytical Chemistry. - : AMER CHEMICAL SOC. - 0003-2700 .- 1520-6882. ; 91:9, s. 6180-6189
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Tidskriftsartikel (refereegranskat)abstract
- A newly developed microscale protocol for profiling serum O-glycans has been validated here with multiple serum samples obtained from different cohorts of colorectal cancer patients. The simultaneous cleavage and permethylation steps in this procedure preserve the integrity of released minor O-glycans, so that 39 O-linked oligosaccharides could be reliably recorded in a profile. This is far more detected components than shown in any previous studies. The analytical results were further subjected to a battery of statistical tests. Our O-glycan compositions compare favorably with the previous results obtained with solid tumors and cancer cell lines, suggesting that smaller circulatory mucins protruding into the blood circulation may be one source of O-glycans that we observe in the serum samples. While the control vs cancer statistical comparisons generally agree with the expected glycosylation trends, the comparisons of male vs female subjects have led to some surprising results for which we do not have a ready explanation due to lack of any literature describing hormonal control of O-glycosylation. Our results thus underscore the necessity of applying new analytical technologies to clinically interesting sample sets.
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| 3. |
- Struwe, Weston B, et al.
(författare)
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The minimum information required for a glycomics experiment (MIRAGE) project: sample preparation guidelines for reliable reporting of glycomics datasets.
- 2016
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 26:9, s. 907-910
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Tidskriftsartikel (refereegranskat)abstract
- Theminimum information required for a glycomics experiment (MIRAGE) project was established in 2011 to provide guidelines to aid in data reporting from all types of experiments in glycomics research including mass spectrometry (MS), liquid chromatography, glycan arrays, data handling and sample preparation. MIRAGE is a concerted effort of the wider glycomics community that considers the adaptation of reporting guidelines as an important step towards critical evaluation and dissemination of datasets as well as broadening of experimental techniques worldwide. The MIRAGE Commission published reporting guidelines for MS data and here we outline guidelines for sample preparation. The sample preparation guidelines include all aspects of sample generation, purification and modification from biological and/or synthetic carbohydrate material. The application of MIRAGE sample preparation guidelines will lead to improved recording of experimental protocols and reporting of understandable and reproducible glycomics datasets.
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| 4. |
- Wada, Yoshinao, et al.
(författare)
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Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.
- 2010
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Ingår i: Molecular & cellular proteomics. - 1535-9484. ; 9:4, s. 719-727
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Tidskriftsartikel (refereegranskat)abstract
- The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.
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