Biomarker discovery by automatic annotation of N-glycan species in MALDI-TOF-TOF spectra

1. Biomarker discovery by automatic annotation of N-glycan species in MALDI-TOF-TOF spectra Chuan-Yih, Yu 2010-4-8 Capstone

2. Introduction • Genomics -> genomes • Proteomics -> proteins • Glycomics -> glycomes • Post-Translation Modification (PTM) • Nitrosylation • Phosphorylation • Glycosolation • 50% of all eukaryotic proteins are glycosylated1 1.Apweiler, R., H. Hermjakob, and N. Sharon, On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.BiochimBiophysActa, 1999. 1473(1): p. 4-8

3. Glycoprotein • Protein glycosylation • N-linked glycosylation • Core structure – 2 GlcNac + 3 Man • Asn-X-Ser or Asn-X-Thr, X can be any but Pro • Glycosylation before folding • O-linked glycosylation • Core structures • Serine or Threonine • Glycosylation after folding

4. Monosaccharides • Building blocks • Diverse linage • Three types N-linked glycan • High mannose • Complex • Hybrid • 412 combinations ->7,000 structures1 1.Krambeck, F.J. and M.J. Betenbaugh, A mathematical model of N-linked glycosylation.BiotechnolBioeng, 2005. 92(6): p. 711-28. Graphs: Varki, A., Essentials of glycobiology. 2nd ed. 2009, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. xxix, 784 p

5. Mass Spectrometry • Wright scale of molecular • Ion Source • Electrosprayionization (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. MALDI-TOF-TOF Graph:MALDI-TOF Mass Analysis. (2008, 11 16). Retrieved May 2, 2009, from The Protein Facility of the Iowa State University Office of Biotechnology www.protein.iastate.edu/maldi.html

7. Problem • Isotope pattern overlap • Permethylated, Add Sodium • 2 GlcNac + 9 Man = 2,396.18 • 7 GlcNac + 3 Man = 2,397.22 • High-throughput glycans screening • Find significant differences between groups of sample http://en.wikipedia.org/wiki/Carbon Graphs: Isotope Pattern Calculator v4.0 http://yanjunhua.tripod.com/pattern.htm

8. Major Features • Glycans profile correlation • Report scores for non-overlap and overlap profile • Glycans examination • Glycan profiling comparison • Report significant glycan between groups • Glycans biomarker discovery

9. Glycans Profile Correlation • For each glycan combination • 412 different glycan combinations • Generate a theoretical isotope pattern • Calculate the correlation for following cases • Glycans • Glycans + Glycans, linear combination applied • Glycans + Unknown, linear combination applied • Mercury algorithm1 1.Rockwood, A., S. Van Orden, and R. Smith, Rapid Calculation of Isotope Distributions. Analytical Chemistry, 1995. 67: p. 2699-2704.

10. Three Cases Glycans Experiment spectrum Glycans α β Unknown Score α β

11. Glycan Profiling Comparison • Multiple spectra comparison • Biomarker discovery • Given spectrum with several conditions • Find distinct glycans between samples HCC: Hepatocellular Carcinoma ( Cancer of liver) CLD: Chronic liver disease Graph: Ressom, H.W., et al., Analysis of MALDI-TOF mass spectrometry data for discovery of peptide and glycan biomarkers of hepatocellular carcinoma. J Proteome Res, 2008. 7(2): p. 603-10.

12. Concept Remove the least significant component. Repeat until all the score above threshold. 70% identical with a cutoff at 0.5 Health spectra (H1, H2, H3…Hk) Disease spectra (D1, D2, D3…Dk) 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

13. Multi N-Glycan • Software Requirement • .netframework 2.0 using C# • C++ runtime • R • Thermo Scientific Xcalibur • Input • Spectrum • Plain text (Peak list) • mzXML1 • RAW ( instrument raw file) • Glycans list • CSV file 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.

14. Software Interface

15. Software Interface Biomarker discovery setting Html result export

16. Result Not present in original composition file Filtered out

17. Result

18. References • Apweiler, R., H. Hermjakob, and N. Sharon, On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.BiochimBiophysActa, 1999. 1473(1): p. 4-8. • 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. • Krambeck, F.J. and M.J. Betenbaugh, A mathematical model of N-linked glycosylation.BiotechnolBioeng, 2005. 92(6): p. 711-28. • 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. • Ressom, H.W., et al., Analysis of MALDI-TOF mass spectrometry data for discovery of peptide and glycan biomarkers of hepatocellular carcinoma. J Proteome Res, 2008. 7(2): p. 603-10. • Rockwood, A., S. Van Orden, and R. Smith, Rapid Calculation of Isotope Distributions. Analytical Chemistry, 1995. 67: p. 2699-2704. • Tang, Z., et al., Identification of N-glycan serum markers associated with hepatocellular carcinoma from mass spectrometry data. J Proteome Res, 2010. 9(1): p. 104-12. • Varki, A., Essentials of glycobiology. 2nd ed. 2009, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. xxix, 784 p.

19. Acknowledge • Advisor: Prof. Haixu Tang • Co-worker: AnoopMayampurath • Collaborator: YehiaMechref, Department of Chemistry • This work will present in 26th May, 58thASMS Conference Salt Lake City, Utahand submit to the Bioinformatics Application Notes.

20. Thank You