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 

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Outline

• Background

• Problem definition and goals

• Implementation of Multi N-Glycan

• Results

• Future work

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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 glycosylated1

[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, 1999. 1473(1): p. 4-8

http://yahoo.brand.edgar-online.com/EFX_dll/EDGARpro.dll?FetchFilingHTML1?SessionID=WD8AC7y2l3h1FMr&ID=5101862

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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

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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

Name Molecular formula/ Structure

Mannose (Man) C6H12O6

Galactose (Gal) C6H12O6

Fucose (Fuc) C6H12O5

GlcNac C8H15NO6

NeuNAC C11H19NO9

NeuNGC C11H19NO10

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Analytical strategies for analyzing glycans

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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

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Mass Spectrometry Spectrum

Isotopic envelope

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N-Glycan Profiling

• Given a MS spectrum screen which glycans present in this spectrum (annotation) and how abundance it is (quantification)

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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.htm http://en.wikipedia.org/wiki/Carbon

2 GlcNac + 9 Man = 2374.5960 7 GlcNac + 3 Man = 2375.63

Mass % Mass %

2371 0.0

2372 84.3 2372 0.0

2373 100.0 2373 82.4

2374 68.5 2374 100.0

2375 34.3 2375 68.8

2376 13.9 2376 34.4

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Problem Definition

7 GlcNac + 3 Man = 2375.632 GlcNac + 9 Man = 2374.59602 GlcNac + 9 Man = 2374.5960

？Unknown2 GlcNac + 9 Man = 2374.5960

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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

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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 algorithm2 - 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.

1.Krambeck, F.J. and M.J. Betenbaugh, A mathematical model of N-linked glycosylation. Biotechnol Bioeng, 2005. 92(6): p. 711-28.

Three scenarios

Experimental isotope envelope

Glycan CorrelationScore

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Theoretical isotope envelope

0.2

0.8

0.6

α

Glycan

β

α

Unknown

β

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Glycan Profiles

• Decompose the abundance for two glycans with overlapping isotopic envelopes

α

Glycans

β

GlycansExperimental

isotope envelope

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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.

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Approach

Health spectra(H1, H2, H3…Hk)

Disease spectra(D1, D2, D3…Dk)

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

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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), mzXML1,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.

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Software Interface

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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

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Software screenshot

Html result export

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Software screenshot

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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

1.Zhiqun T., et al., Identification of N-Glycan Serum Markers Associated with Hepatocellular Carcinoma from Mass Spectrometry Data. J Proteome Res, 2009

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Result Derived from The Paper

Filtered out

Can’t find the glycan structure neither in my list nor CFG database

Low c

orre

latio

n

scor

e

Ove

rlap

with 2

192

Zhiqun T., et al., Identification of N-Glycan Serum Markers Associated with Hepatocellular Carcinoma from Mass Spectrometry Data. J Proteome Res, 2009

Identified

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Result Derived from Multi N-Glycan

Confirmed resultDistinct glycan

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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

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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 26th 2010, 58th 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.

Thank You