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Computational Methods for
Biomarker Discovery in Proteomics
and Glycomics
Vijetha Vemulapalli
School of Informatics
Indiana University
Capstone Advisor: Dr. Haixu Tang
What are Biomarkers?
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
 Substances present in increased or decreased
amounts in body fluids or tissues that indicate
exposure, disease or susceptibility to disease.
Some Uses of Biomarkers
• Problem Definition
• Background
• LC-MS
• Method
• Results
 Biomarkers are increasingly being used for the
following purposes:
• Prognosis / Diagnosis of disease
• Monitoring response to medication
• CE
• Method
• Results
• Acknowledgements
• References
 With high sensitivity and throughput, proteomics
and glycomics is capable of identifying many potential
biomarkers simultaneously.
More on Biomarkers
• Problem Definition
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
Quantity
• Background
10
9
8
7
6
5
4
3
2
1
0
Normal
Diseased
A
B
C
D E F G H
Substance
I
J
 A lot of times biomarkers have not been identified
clearly. But based on the signature pattern of glycans
and proteins, samples can be classified as healthy and
diseased.
What is Proteomics?
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
 Proteins: A chain of amino acids
including hormones, enzymes and
antibodies.
 Proteome: All the proteins in a cell or
bodily fluid at a given point of time under
certain conditions.
• Method
• Results
• Acknowledgements
• References
Proteomics: Proteomics is the study of
proteins and proteomes using highthroughput technology.
http://parasol.tamu.edu/groups/amatogroup/foldingserver/images/proteinL.gif
http://biology.clc.uc.edu/graphics/bio104/cell.jpg
What is Glycomics?
• Problem Definition
• Background
• LC-MS Method
• Results
 Glycoproteins: Proteins with
attached polysaccharides .
 Glycans: Polysaccharide chain
attached to a protein
• CE
• Method
• Results
• Acknowledgements
• References
 Glycome: The entire set of
glycans that are present in a cell
or a bodily fluid at a certain point
of time under certain conditions.
 Glycomics: Study of structure and function of
oligosaccharides in a cell or organism.
http://www.glyfdis.org/images/bg_image.jpg
High Throughput Technologies to
Identify Biomarkers
• Problem Definition
• Background
• LC-MS
• Method
• Results
Genome Scale
Scanning
Micro - arrays
Transcriptome
level
• CE
• Method
Proteomics
• Results
• Acknowledgements
• References
Genome
level
Proteome
level
Glycomics
Glycome
level
http://phy.asu.edu/phy598-bio/D4%20Notes%2006_files/image002.jpg
Why the Focus on Proteomics and
Glycomics?
• Problem Definition
 Information content
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
Genome
Transcriptome
Transcriptome
Proteome
• Acknowledgements
• References
Static
Glycome
Dynamic
Biomarker Discovery using
Proteomics
Liquid Chromatography / Mass
Spectrometry (LC/MS)
• Problem Definition
• Background
• LC-MS
Protein
sample
Liquid
Chromatography
Mass
Spectrometry
Data
• Method
• Results
 Why LC/MS for analysis of proteomes?
• CE
• Method
• Results
• Acknowledgements
• References
• LC spreads complexity of the sample over time.
• MS identifies ions based on their mass/charge value.
 Software exists currently to identify proteins in a
sample using data from a LC-MS experiment.
Liquid Chromatography (LC)
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
http://wwwlb.aub.edu.lb/~webcrsl/high_p3.jpg
 Liquid Chromatography is a technique that
separates ions or molecules dissolved in a solvent
based on size of the ion/molecule, adsorption, ionexchange or other similar characteristics.
What is Mass Spectrometer?
• Problem Definition
• Background
 Mass Spectrometry (MS) is an instrument that
identifies ions based on their mass-to-charge ratio.
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
Source: http://www.chemguide.co.uk/analysis/masspec/howitworks.html & http://www.bmms.uu.se/ltq-ft.htm
Visualization of LC/MS Data : 2D
Map
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
How Do We Find Biomarkers From
LC-MS Data?
• Problem Definition
• Background
Liquid
Chromatography
Protein
sample
Mass
Spectrometry
Data
• LC-MS
• Method
• Results
• CE
Identified Proteins
and Peptides
Identification
software
• Method
• Results
• Acknowledgements
• References
MS View
Quantities of peptides identified from the
sample
How Do We Find Biomarkers From
LC-MS Data? Continued…
• Problem Definition
• LC-MS
• Method
Quantification 1
Sample 2
Quantification 2
Sample 3
Quantification 3
MSView
• Results
• CE
• Method
• Results
• Acknowledgements
• References
Sample N
Quantification N
Analyze to find Biomarkers
• Background
Sample 1
MSView
• Problem Definition
MSView
• Background
• LC-MS
• Method
• Results
Components
Visualization
• CE
Relative
Quantification
• Method
• Results
• Acknowledgements
• References
Purpose
Visual comparison
/Analysis
Further
analysis for
Biomarker
Discovery
Extracted Ion Chromatogram (XIC)
• Problem Definition
• Background
• LC-MS
 Chromatogram created by plotting the intensity of
the signal observed at a chosen m/z value in a series of
mass spectra recorded as a function of retention time.
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
Source: http://www.lcpackings.com/applications/Probot/images/dual_fract04B.png
Visualization: XIC
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
Relative Quantification using Peptide
Identification Results
• Problem Definition
Data from LC-MS
experiment
• Background
Identification of
peptides
• LC-MS
• Results
• CE
• Method
• Results
MS View
• Method
Extracted Ion Chromatogram of peptide
Peak selection
• Acknowledgements
• References
Area calculation
Max  Min   Max
Quantification: Peak Selection
Algorithm
• Problem Definition
Selecting
After
Smoothing:
peaks:
Actual
data:
Selecting
local maxima and minima
• Background
• LC-MS
Minima
Minima
Maxima
Maxima
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
Max  Min   Max
Quantification: Sample Results
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
Biomarker Discovery using
Glycomics
How does Capillary Electrophoresis
(CE) work?
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
http://faculty.washington.edu/dovichi/UBUBTpage/research/Methods/CEintro/ceintro.GIF&imgrefurl=http://faculty.washington.edu/dovichi/UBUBTpage/research/Methods/CEintro/CE_LIF.html&h=531&w=6
84&sz=25&hl=en&start=3&um=1&tbnid=_JDf4X3dJn170M:&tbnh=108&tbnw=139&prev=/images%3Fq%3Dcapillary%2Belectrophoresis%26svnum%3D10%26um%3D1%26hl%3Den
What does the data look like?
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
Samples from different CE experiments:
Biomarker Discovery using Glycomics
– Overview
• Problem Definition
Data from different samples
• Background
• LC-MS
• Results
• CE
• Method
• Results
CE Analyze
• Method
Mapping areas corresponding the same glycan
from different samples
Quantification of mapped peaks
• Acknowledgements
• References
Analysis of quantification for identifying Biomarkers
Direct Comparison: Dynamic Time
Warping (DTW)
• Problem Definition
• Background
• LC-MS
• Method
• Results
 DTW
algorithm
aligns two time series
having similar curves
but
are
skewed
differently over time.
• CE
• Method
• Results
• Acknowledgements
• References
Time
Source: http://db-www.aist-nara.ac.jp/theme/bioinfo_kenji-h_dtw.png
Direct Comparison: DTW continued…
• Background
 Sakoe-Chuba Band is used to reduce time & space
complexity.
• LC-MS
 Parameters used in DTW:
• Problem Definition
• Method
• Results
- Band width
- Peak extention penalty
- Difference in peak intensities.
• CE
• Method
- Difference in peak direction
• Results
• Acknowledgements
• References
Stan Aslvador and Philip Chan. FastDTW:Toward Accurate Dynamic Time Warping in Linear Time and Space, KDD Workshop on Mining
Temporal and Sequential Data, 2004
Method: Dynamic Time Warping
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
Align
to consensus sample
Consensus
Align next sample to
Sample
consensus sample
Method continued…
• Problem Definition
• Background
Unaligned sample
• LC-MS
• Method
Corresponding peaks
Aligned sample
• Results
Corresponding peaks
• CE
• Method
• Results
• Acknowledgements
• References
Calculate Area
Peak 1
Results
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
Corresponding peaks
Summary
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References
 Proteomics - MSView
LC-MS data
Identified Peptides
Quantification results for
Biomarker Discovery
Glycomics - CE Analyze
CE Data
Quantification results for
Biomarker Discovery
Acknowledgements
• Problem Definition
• Background
• LC-MS
• Method
• Results
• CE
Dr. Haixu Tang
- My advisor
Dr. Randy J.Arnold
Dr. Milos Novotny
Dr. Sun Kim
Dr. Stephen J. Valentine
Dr. Yehia Mechref
Dr. David E.Clemmer
Dr. Jeong-Hyeon Choi
Yin Wu
Manolo D.Plasencia
• Method
• Results
• Acknowledgements
• References
School of Informatics
Funding:
NIH/NCRR
MetaCyt Initiative @ Indiana University
References
• Problem Definition
[1] Higgs, R.E., Knierman, M.D., Gelfanova, V., Butle,r J.P. and Hale,
• Background
J.E. (2005) Comprehensive label-free method for the relative
quantification of proteins from biological samples. J. Proteome Res., 4,
1442-1450.
[2] Linsen, L., Locherbach, J., Berth, M., Becher, D. and Bernhardy, J.
(2006) Visual Analysis of Gel-Free Proteome Data. IEEE Transactions on
Visualization and Computer Graphics,12, 497-508.
[3] Prakash, A., Mallick, P., Whiteaker, J., Zhang, H., Paulovich, A.,
Flory, M., Lee, H., Aebersold, R., and Schwikowski, B. (2006) Signal
maps for mass spectrometry-based comparative proteomics. Mol. Cell.
Proteomics 5, 423 –432
[4] Leptos, K. C., Sarracino, D. A., Jaffe, J. D., Krastins, B., and Church,
G. M. (2006) MapQuant: open-source software for large-scale protein
quantification. Proteomics 6, 1770 –1782
[5] Aebersold, R., and Mann, M. (2003) Mass spectrometry-based
proteomics. Nature 422, 198 –207
• LC-MS
• Method
• Results
• CE
• Method
• Results
• Acknowledgements
• References