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

Outline Introduction –Glycoprotein, Monosaccharides, N-linked glycosylation, and Mass Spectrometry Problem set Goals MultiNGlycan Result Future works 1

Introduction Post-Translation Modification (PTM) –An enzyme-catalyzed change after synthesized –Acetylation, Cleavage, Glycosylation, Methylation, Phosphorylation, and Prenylation 50% of all eukaryotic proteins are glycosylated 1 [Apweiler, et al.] 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, (1): p

Glycosylation N-linked glycosylation –Core structure – 2 GlcNac + 3 Man –Asn-X-Ser or Asn-X-Thr, X can be any but Pro (glycosylation sequon) –Glycosylation before folding O-linked glycosylation –Many different core structures –Serine or Threonine –Glycosylation after folding 3

N-linked glycosylation Tree structure Monosaccharides- building blocks of polysaccharide chain Diverse linage – 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

Mass Spectrometry Wright scale of molecular 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 5

MALDI-TOF-TOF Matrix-assisted laser desorption/ionization Time of flight (TOF) 6 Graph:MALDI-TOF Mass Analysis. (2008, 11 16). Retrieved May 2, 2009, from The Protein Facility of the Iowa State University Office of Biotechnology

Problem Sets Glycopeptide isotope pattern overlap Graphs: Isotope Pattern Calculator v GlcNac + 9 Man = GlcNac + 3 Man = Mass% %

Problem Sets High-throughput glycans profiling 8

Goals Glycans profile correlation –Report scores for non-overlap and overlap profile –Glycans examination Glycan profile comparison –Report significant glycan between groups –Glycans biomarker discovery 9

Glycans Profile Correlation For each glycan combination –412 different glycan combinations [Krambeck, et al. ] 1 –Generate a theoretical isotope pattern –Calculate the correlation for following cases 1.Glycans 2.Glycans + Glycans, linear combination applied 3.Glycans + Unknown, linear combination applied Mercury algorithm 2 –Generate the unknown isotope pattern 2.Rockwood, A., S. Van Orden, and R. Smith, Rapid Calculation of Isotope Distributions. Analytical Chemistry, : p Krambeck, F.J. and M.J. Betenbaugh, A mathematical model of N-linked glycosylation. Biotechnol Bioeng, (6): p

Three Cases Experiment spectrum Glycans α α Unknown Score 11 Theoretical isotope pattern β β

Glycan Profile Comparison Multiple spectra comparison Biomarker discovery –Given spectrum with several conditions –Find distinct glycans between samples 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, (2): p HCC: Hepatocellular Carcinoma ( Cancer of liver) CLD: Chronic liver disease 12

Concept 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, (2): p. RESEARCH % identical with a cutoff at

Multi N-Glycan Software Requirements –.net framework 2.0 using C# –C++ runtime –R –Thermo Scientific Xcalibur Input –Spectrum Plain text (Peak list), mzXML 1,RAW (Thermo Scientific raw file) –Glycans list CSV file (User-defined) 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, (11): p

Software Interface 15

Software features Signal preprocessing provided –Subtracting background –Smoothing peak –Tolerating Mass Spectrometry accuracy Flexible parameters incorporate actual experiment Useful tools provides –Isotope pattern generator Content rich output, multi-format supports –csv, text, html 16

Software screenshot 17 Html result export

Software screenshot 18

Result Data set –Liver Cancer : 73 individuals –Health: 78 individuals 412 glycan structures are tested Glycan criterion –Correlation score cut off < 0.5 –Present in 30% of total spectra 19 Zhiqun T., et al., Identification of N-Glycan Serum Markers Associated with Hepatocellular Carcinoma from Mass Spectrometry Data. J Proteome Res, 2009 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, (2): p Anoop M., Chuan-Yih Y., A Multi-PCA Approach to Glycan Biomarker Discovery using Mass Spectrometry Profile Data. I690 project, 2009 Fall

Result Filtered out Can’t find the glycan structure in CFG database 20 Correlation score Overlap with 2192

Result 21

Future Works Test on more clinical samples Verify the correlation between glycan modification which reported by MultiNGlycan with Hepatocellular arcinoma Perform these tasks on O-linked glycan Apply de novo glycan sequencing on reported glycan (ongoing) 22

References Anoop M., Chuan-Yih Y., A Multi-PCA Approach to Glycan Biomarker Discovery using Mass Spectrometry Profile Data. I690 project, 2009 Fall 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, (1): p Dalit Shental-Bechor and Yaakov Levy, Effect of glycosylation on protein folding: A close look at thermodynamic stabilization, PNAS June 11, 2008 Hastie, T., et al., ‘Gene shaving’ as a method for identifying distinct sets of genes with similar expression patterns. Genome Biol, (2): p. RESEARCH0003. Krambeck, F.J. and M.J. Betenbaugh, A mathematical model of N-linked glycosylation. Biotechnol Bioeng, (6): p Pedrioli, P., et al., A Common Open Representation of Mass Spectrometry Data and its Application in a Proteomics Research Environment. Nature Biotechnology, (11): p 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, (2): p Rockwood, A., S. Van Orden, and R. Smith, Rapid Calculation of Isotope Distributions. Analytical Chemistry, : p Zhiqun, T., et al., Identification of N-glycan serum markers associated with hepatocellular carcinoma from mass spectrometry data. J Proteome Res, (1): p Varki, A., Essentials of glycobiology. 2nd ed. 2009, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. xxix, 784 p. 23

Acknowledge Advisor: Prof. Haixu Tang Co-worker: Anoop Mayampurath Collaborator: Yehia Mechref, Department of Chemistry COL Lab members This work will present in 26 th May, 58 th ASMS Conference Salt Lake City, Utah and submit to the Bioinformatics Application Notes. 24

Thank You