1. Automatic annotation of N-glycans in MALDI-TOF spectra for rapid glycan profiling and comparison Chuan-Yih, Yu 2010.05.14 Capstone Presentation Advisor: Prof. Haixu Tang Indiana University Bloomington School of Informatics and Computing

2. Outline Background Problem definition and goals Implementation of Multi N-Glycan Results Future work 1

3. Background Post-Translation Modification (PTM) –Enzyme-catalyzed protein modification after protein synthesized –Acetylation, Glycosylation, Methylation, Phosphorylation, Prenylation, and etc. >50% of all eukaryotic proteins are glycosylated 1 [Apweiler, et al.] 2 1.Apweiler, R., H. Hermjakob, and N. Sharon, On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta, 1999. 1473(1): p. 4-8 http://yahoo.brand.edgar-online.com/EFX_dll/EDGARpro.dll?FetchFilingHTML1?SessionID=WD8AC7y2l3h1FMr&ID=5101862

4. Glycosylation Attachment of a glycan(sugar) to the peptide chain N-linked glycosylation –Nitrogen link to Asn –Asn-X-Ser(NXS) or Asn-X-Thr(NXT), X can be any but Pro (glycosylation sequon) –Core structure – 2 GlcNac + 3 Man –Glycosylation while folding O-linked glycosylation –Many different core structures –Serine or Threonine –Glycosylation after folding 3

5. N-linked glycosylation Tree structure Monosaccharides- building blocks of polysaccharide chain Diverse linkage – at most four branches Three types of N-linked glycan tree –High mannose –Complex –Hybrid Graphs: Varki, A., Essentials of glycobiology. 2nd ed. 2009, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. xxix, 784 p NameMolecular formula/ Structure Mannose (Man)C 6 H 12 O 6 Galactose (Gal)C 6 H 12 O 6 Fucose (Fuc)C 6 H 12 O 5 GlcNacC 8 H 15 NO 6 NeuNACC 11 H 19 NO 9 NeuNGCC 11 H 19 NO 10 4

6. Analytical strategies for analyzing glycans 5

7. Mass Spectrometry Wright scale of molecular High throughput, High accuracy, High sensitivity Ion Source –Electrospray ionization (ESI) –Matrix-assisted laser desorption/ionization (MALDI) Mass Analyzer –Time of flight (TOF) –Quadrupole –Fourier transform mass spectrometry (FTMS) Detector –Charge induced or the current produced 6

8. Mass Spectrometry Spectrum 7 Isotopic envelope

9. N-Glycan Profiling Given a MS spectrum screen which glycans present in this spectrum (annotation) and how abundance it is (quantification) 8

10. Problem Definition Glycan isotope envelope –Isotope present in the natural world different numbers of neutrons Graphs: Isotope Pattern Calculator v4.0 http://yanjunhua.tripod.com/pattern.htmhttp://yanjunhua.tripod.com/pattern.htm http://en.wikipedia.org/wiki/Carbon 9 2 GlcNac + 9 Man = 2374.59607 GlcNac + 3 Man = 2375.63 Mass% % 23710.0 237284.323720.0 2373100.0237382.4 237468.52374100.0 237534.3237568.8 237613.9237634.4

11. Problem Definition 10 7 GlcNac + 3 Man = 2375.63 2 GlcNac + 9 Man = 2374.5960 ？ Unknown 2 GlcNac + 9 Man = 2374.5960

12. Goals Annotation of N-glycan –Decompose observed isotopic envelopes into non-overlapping and overlapping isotopic envelopes of glycan –Quantify the relative abundance of glycan Glycan profile comparison –Report glycans that show significant different abundance between groups of samples –Discover glycan biomarkers 11

13. Glycans Annotation For each glycan ( i.e. monosaccharides composition) –412 different glycans [Krambeck, et al. ] 1 –Generate a theoretical isotope envelope –Calculate the correlation between the theoretical and observed isotope envelopes for each of following scenarios 1.Glycans 2.Glycans + Glycans, linear fitting applied 3.Glycans + Unknown, linear fitting applied –Mercury algorithm 2 - generate the unknown isotope envelopes 2.Rockwood, A., S. Van Orden, and R. Smith, Rapid Calculation of Isotope Distributions. Analytical Chemistry, 1995. 67: p. 2699-2704. 12 1.Krambeck, F.J. and M.J. Betenbaugh, A mathematical model of N-linked glycosylation. Biotechnol Bioeng, 2005. 92(6): p. 711-28.

14. Three scenarios Experimental isotope envelope Glycan Correlation Score 13 Theoretical isotope envelope 0.2 0.8 0.6 α Glycan β α Unknown β

15. Glycan Profiles Decompose the abundance for two glycans with overlapping isotopic envelopes 14 α Glycans β Experimental isotope envelope

16. Glycan Profile Comparison Comparison of glycan abundances in multiple samples Biomarker discovery –Given glycan spectra from multiple samples under different (e.g. disease vs. health) conditions –Goal: To find glycans with distinct abundances between samples Z Kyselova, Y. Mechref, M. M. Al Bataineh, L. E. Dobrolecki, R. J. Hickey, J. Vinson, C. J. Sweeney, and M. V. Novotny. Alterations in the serum glycome due to metastatic prostate cancer. Journal of Proteome Research, 6:18221832, 2007. 15

17. Approach Health spectra (H 1, H 2, H 3 …H k ) Disease spectra (D 1, D 2, D 3 …D k ) Remove the least significant component. Repeat until all the score above threshold. 1.Hastie, T., et al., 'Gene shaving' as a method for identifying distinct sets of genes with similar expression patterns. Genome Biol, 2000. 1(2): p. RESEARCH0003 70% identical with a cutoff at 0.5 16

18. Implementation of Multi N-Glycan Software Requirements –.net framework 2.0 using C# –C++ runtime –[R] for PCA analysis –Thermo Scientific Xcalibur Input –Spectrum File format: Plain text (Peak list), mzXML 1,RAW file (Thermo Scientific raw file) –N-Glycans list CSV file (User-defined); default define by [Krambeck, et al. ] Output –List of glycans with scores 1.Pedrioli, P., et al., A Common Open Representation of Mass Spectrometry Data and its Application in a Proteomics Research Environment. Nature Biotechnology, 2004. 22(11): p. 1459-1466. 17

19. Software Interface 18

20. Software features Signal preprocessing provided –Subtracting background –Smoothing and picking peaks –Tolerating mass accuracy Flexible parameters incorporate actual experiment Isotope envelopes generator Content rich output, supporting multiple formats –csv, text, html 19

21. Software screenshot 20 Html result export

22. Software screenshot 21

23. Result Data set [ Zhiqun T., et al] –Liver Cancer : 73 individuals –Health: 78 individuals 412 N-glycan are used Parameters –Correlation score < 0.5 will be discarded. –Present in >30% of all samples 22 1.Zhiqun T., et al., Identification of N-Glycan Serum Markers Associated with Hepatocellular Carcinoma from Mass Spectrometry Data. J Proteome Res, 2009

24. Result Derived from The Paper Filtered out 23 Low correlation score Overlap with 2192 Zhiqun T., et al., Identification of N-Glycan Serum Markers Associated with Hepatocellular Carcinoma from Mass Spectrometry Data. J Proteome Res, 2009 Identified

25. Result Derived from Multi N-Glycan 24 Confirmed result Distinct glycan

26. Future Work Test on more clinical samples Extend to O-glycan profiling Apply de novo glycan sequencing on reported glycan (ongoing) Connect reported glycans to glycan research literatures 25

27. Acknowledge Advisor: Prof. Haixu Tang Co-worker: Anoop Mayampurath Collaborator: Yehia Mechref, Department of Chemistry COL Lab members This work will be presented on May 26 th 2010, 58 th ASMS Conference Salt Lake City, Utah; and will be submitted to the Bioinformatics. This work is funded by NCI/NIH grant number 1 U01 A128535-01. 26

28. Thank You