Abstract
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.
Abbreviations
- 2AA:
-
2-aminobenzoic acid
- AAL:
-
Aleuria aurantia lectin
- CDG:
-
Congenital disorders of glycosylation
- ConA:
-
Concanavalin A
- DEAE:
-
Diethylaminoethyl
- EIC:
-
Extracted ion chromatogram
- ESI:
-
Electrospray ionization
- ETD:
-
Electron-transfer dissociation
- HCD:
-
High-energy collision-induced dissociation
- HGPI:
-
Human Disease Glycomics/Proteome Initiative
- HPLC:
-
High performance liquid chromatography
- HUPO:
-
Human Proteome Organization
- Ig:
-
Immunoglobulin
- LC:
-
Liquid chromatography
- MALDI:
-
Matrix-assisted laser desorption ionization
- MIRAGE:
-
Minimum information required for a glycomics experiment
- MS:
-
Mass spectrometry
- PVDF:
-
Polyvinylidene fluoride
- PA:
-
Pyridylaminated
- PGC:
-
Porous graphitic carbon
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Acknowledgments
Authors thank Drs. Yuzuru Ikehara and Ta-Wei Liu, and Ms. Azusa Tomioka of the National Institute of Advanced Industrial Science and Technology (AIST) for preparation and distribution of the samples. PA and MI acknowledge funding from the National Institutes of Health (NIH)-funded Research Resource for Biomedical Glycomics (P41GM10349010) to the Complex Carbohydrate Research Center. DK acknowledges funding from the European Union (Seventh Framework Program “Glycoproteomics”, grant number PCIG09-GA-2011-293847). NP, MT-A acknowledge funding from the Australian Research Council. NGK would like to acknowledge the financial contribution from Swedish Research Council (grant no 342-2004-4434 and no 621-2013-5895). All the authors would like to acknowledge the tremendous contribution that Azumi Takahashi of the AIST has made in assembling this paper and bringing it to fruition. This paper is dedicated to the memory of Prof. Kazuaki Kakehi of Kinki University who died on May 28, 2014 and we acknowledge his contribution to this project.
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Kazuaki Kakehi deceased in 2014
Hiromi Ito and Hiroyuki Kaji contributed equally to this work.
Hisashi Narimatsu served as the chair of the Human Disease Glycomics/Proteome Initiative, HUPO.
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Ito, H., Kaji, H., Togayachi, A. et al. Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines. Glycoconj J 33, 405–415 (2016). https://doi.org/10.1007/s10719-015-9625-3
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DOI: https://doi.org/10.1007/s10719-015-9625-3