1. Carbonyl-Reactive Tandem Mass Tags for the Proteome-Wide Quantification of N-Linked Glycans Hannes Hahne, Patrick Neubert, Karsten Kuhn, Chris Etienne, Ryan Bomgarden, John C. Rogers, and Bernhard Kuster 2015/04/29 Speaker : Guo-Ming Hong Advisor : Dr. Pang-Hung Hsu Analytical Chemistry 2012 84 (8), 3716-3724

3. Introduction

4. Glycosylation is one of the most abundant post- translational modifications : Cell−cell interactions Cellular recognition, proliferation and development Immune response Protein stability

5. N-glycan: Nature Reviews Microbiology 5, 583-597 (August 2007) high-mannose typecomplex-typehybrid type

6. More recently, MS-based quantification has become increasingly popular. Glycan labeling : 1.Permethylation 2.Reductive amination, hydrazone, or oxime formation.

7. Permethylation has long been used because : 1.Stabilization of acidic glycans 2.Increase overall MS signal strength 3.Separate the glycans by reversed phase chromatography

8. Tandem mass tags (TMTs) have been originally developed for the isobaric labeling and quantification. https://www.lifetechnologies.com/order/catalog/product/90063

9. Glycan labeling at reducing end + TMT

10. MATERIALS AND METHODS

11. Standard Glycans : Chicken egg ovalbumin bovine α 1 -acid glycoprotein disialylacto-N-tetraose (DSLNT) Cell Line : SW480 SW620 TMT-Labeling Hydrazide TMT Aminooxy TMT PNGase F methyl iodide MALDI-TOF/TOF

12. https://worldwide.promega.com/products/mass-spectrometry/proteases-and-surfactants/pngase-f/?activeTab=1 PNGase F Cleavage Site :

13. TMT-Labeling :

14. MALDI-TOF : http://www.igb.fraunhofer.de/de/kompetenzen/grenzflaechentechnik/oberflaechenanalytik/ausstattung/maldi-tof.html MALDI-TOF/TOF :

15. RESULTS AND DISCUSSION

16. Novel Carbonyl-Reactive Tandem Mass Tags for Glycan Quantification TMT Labeling Efficiency and Stabilization of Acidic Glycans Quantification of Heavy/Light-Labeled Glycans Quantification of Isobarically Labeled Glycans Quantitative Glycan Profiling of Isogenic Colorectal Cancer Cell Lines Future Directions

17. Novel Carbonyl-Reactive Tandem Mass Tags for Glycan Quantification TMT Labeling Efficiency and Stabilization of Acidic Glycans Quantification of Heavy/Light-Labeled Glycans Quantification of Isobarically Labeled Glycans Quantitative Glycan Profiling of Isogenic Colorectal Cancer Cell Lines Future Directions

18. Glyco-TMT Hydrazide Aminooxy A : hydrazide reagents B : aminooxy reagents

19. Novel Carbonyl-Reactive Tandem Mass Tags for Glycan Quantification TMT Labeling Efficiency and Stabilization of Acidic Glycans Quantification of Heavy/Light-Labeled Glycans Quantification of Isobarically Labeled Glycans Quantitative Glycan Profiling of Isogenic Colorectal Cancer Cell Lines Future Directions

20. quantitative aminooxy TMT labeling was achieved at submillimolar TMT concentration

21. Combined aminooxy TMT-labelling and methylation of a sialylated glycan.

22. Novel Carbonyl-Reactive Tandem Mass Tags for Glycan Quantification TMT Labeling Efficiency and Stabilization of Acidic Glycans Quantification of Heavy/Light-Labeled Glycans Quantification of Isobarically Labeled Glycans Quantitative Glycan Profiling of Isogenic Colorectal Cancer Cell Lines Future Directions

23. Ovalbumin with light (TMT 0 ) and heavy (TMT 6 ) forms of both hydrazide and aminooxy TMT MALDI TOF spectrum of heavy and light labeled glycans

24. Dynamic range and accuracy of hydrazide and aminooxy TMT quantification. N-linked glycans were mixed in known ratios (1:1, 1:5, 1:10, 1:20, and 1:40) Heavy/light labeling with glyco-TMT enables accurate quantification in MALDI MS spectra.

25. Novel Carbonyl-Reactive Tandem Mass Tags for Glycan Quantification TMT Labeling Efficiency and Stabilization of Acidic Glycans Quantification of Heavy/Light-Labeled Glycans Quantification of Isobarically Labeled Glycans Quantitative Glycan Profiling of Isogenic Colorectal Cancer Cell Lines Future Directions

26. MALDI TOF/TOF spectrum of labeled Man 6 GlcNAc 2 (m/z 1720.708, aminooxy TMT 6 -128/131, 1:1) Reporter ion-based quantification of isobarically labeled N- linked ovalbumin glycans.

27. Dynamic range and accuracy of hydrazide and aminooxy TMT quantification

28. Novel Carbonyl-Reactive Tandem Mass Tags for Glycan Quantification TMT Labeling Efficiency and Stabilization of Acidic Glycans Quantification of Heavy/Light-Labeled Glycans Quantification of Isobarically Labeled Glycans Quantitative Glycan Profiling of Isogenic Colorectal Cancer Cell Lines Future Directions

29. Aminooxy TMT 0/6 quantification of neutral and acidic glycans from the colorectal primary tumor cancer cell line SW480 and its isogenic metastatic variant SW620. MALDI MS spectrum of the mixed N-glycan pools.

30. significantly regulated N-glycans. Light and heavy monoisotopic glycan signals are indicated (*) where required. Expanded MALDI spectra for the spiked standard DSLNT and selected unregulated given the potential involvement of cell surface glycosylation in cell migration and invasion of cancer cells.

31. Significantly Differential Glycan Compositions from SW480 and SW620 Cells

32. Deposited in the Gene Expression Atlas indicate a general down- regulation of key enzymes involved in high-mannose trimming in the primary tumor cell line SW480 compared to SW620. mRNA expression data of high-mannose trimming enzymes deposited at Gene Expression Atlas from the study of Jakobsen et al. 2 1 (data source: http://www.ebi.ac.uk/gxa/experiment/E-MEXP-1014/ENSG00000115275?ef=cell_line)

33. Novel Carbonyl-Reactive Tandem Mass Tags for Glycan Quantification TMT Labeling Efficiency and Stabilization of Acidic Glycans Quantification of Heavy/Light-Labeled Glycans Quantification of Isobarically Labeled Glycans Quantitative Glycan Profiling of Isogenic Colorectal Cancer Cell Lines Future Directions

34. To achieve accurate quantification : 1.vendor’s software 2.a standard spreadsheet application

35. Conclusions

36. Carbonyl-reactive TMTs as novel tools for the quantitative analysis of protein glycosylation from single proteins through to complex biological samples such as cancer cell lines. Future work will address the potential of this approach for such applications and an extension to N-glycan profiling by LC−MS which likely offers a more in-depth coverage of the many compositional glycan isomers that exist in nature.

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