The Critical Role of Algorithms in the Interpretation of Multiplexed

Protein Biomarker PanelsStephen TC Wong

Departments of Radiology and Pathology, The Methodist HospitalThe Center for Biotechnology and Informatics, TMH Research Institute,

Weill Cornell Medical College

Common Proteomics Techniques

2D gel analysisKronberg, et al, Clin Chem. 1984

MS (Mass Spectrometry) analysis Blonder, J. et al. Nature Protocols 2007Protein array analysis

Pajak, B. et al. J Physiol Pharmacol. 2005

Sample CollectionSerum/plasmaTissue/bio-fluid

Experimental design

MS Proteomics Operation FlowchartHigh throughput

Sample processingLab robot

Enrichment Fractionation

Albumin depletion

Albumin-bound

Magnetic beads

Mass Spec

BioinformaticsStatistics Algorithms

ImmunoassayValidation

Multiple biomarker

Diagnostic kit

Tissue array

ELISA tests

Protein array

SELDI

MALDI

High resolution

Data preprocessing

Feature selection

Biomarker discovery

Protein identification

Comparison of Proteomics Platforms

Negm et al, TRENDS in Molecular Medicine Vol.8 No.6 June 2002

Comparison of MALDI MS Types

Challenges of Sample Prep

Is blood the best place to look?

Extraordinary dynamic range in blood proteins

Depletion of abundant or dominant plasma proteins is not without risk as these proteins may act as carriers for low-abundance molecules

Variations due to environmental factors: genetic polymorphism, gender, age, ethnicity, life style, dietary influences, diurnal factors, and co-morbidities

Variations arising from experimentation: blood collection, processing time, sample preparation, storage temperature, and variable release of platelet contents.

Current Opinion in Chemical Biology 2008 12:72-77

Challenges for Peptidome

Does the blood ‘peptidome’ reveal the disease?A number of peptides may be missed or wrongly assigned due to experimental limitations related to blood collection, processing, and storage protocols

Current Opinion in Chemical Biology 2008, 12:72-77

Algorithms: Baseline Removal

From: Methods in Molecular Biology, vol.328: new and Emerging proteomic Techniques

Algorithms : Normalization

http://www.mathworks.com/products/demos/bioinfo/massspec_prepro/mspreprodemo_07.png

Normalizes a group of mass spectra

Algorithms : DenoisingWavelet

Estimate noiseProteomics 2005, 5:4107-4117

DOI 10.1002/mass.20072

Algorithms : Peak detection

Mean Max

Doi: 10.1093/bioinformatics/bt254

Proteomics 2006,6:5106-5116

Reversible Jump MCMC Approach of Peak detection for SELDI-MS

Yuan Wang et al, Bioinformatics, 2008

A Bayesian Peak Detection Algorithm for PrOTOF MALDI-MS

Jianqiu Zhang et al, Bioinformatics,2008

Algorithms: Peak alignment

Msignt software

W.Yu et al. Computational Biology and Chemistry, 2006DOI 10.1002/mas.20072

Algorithms: Feature Selection for Biomarker Discovery

T test; F test

Genetic algorithm

Natural inspired

Random forest/random decision tree

Weight vector of SVM

Neural network

Saeys et al, Bioinformatics. 2007

Panel biomarkers using genetic algorithms with Recursive Local Floating Search for feature selection

Xiaobo Zhou et al, Proteomics, in revision, 2008

Algorithms: Classification

KNN

PCA

Decision Tree (DT)− SVM (Support Vector Machine)

− Decision Tree (DT)

− Neural Network

− KNN (K-Nearest Neighbors)

− PCA (Principal Component Analysis)

− LDA, QDA, Logistic Regression

http://www.mirrorservice.org/sites/home.ubalt.edu/ntsbarsh/Business-stat/opre/RiskTree.gifhttp://upload.wikimedia.org/wikipedia/commons/thumb/e/e7/KnnClassification.svg/279px-KnnClassification.svg.png

Algorithms: Protein/Peptide Identification

http://www.scq.ubc.ca/wp-content/uploads/2006/08/Proteomics.gif

http://news.thomasnet.com/images/large/801/801252.jpg

Problems in MS Proteomics

Inconsistent and often irreproducible results due to

different methods and algorithms.

Feature selection is a NP-hard (exponential) problem

Classification: Models selected, parameters chosen,

and overfitting

Difficult to scale up for high throughput protein

identification.

Diverse strategies, algorithms, parameters result in:

Validation via Co-Immunoprecipitation

http://www.piercenet.com/Products/Browse.cfm?fldID=9C471132-0F72-4F39-8DF0-455FB515718F

KIYONAKA et al, Semi-wet peptide/protein array using supramolecular hydrogel, nature materials, 2004

Validation via Protein Array

Validation via Tissue Array

• Validates genes or proteins identified by genomic or proteomic analysis

• Validates new biomarkers of tumor progression

Validation via Nanoparticles

http://www.nsti.org/news/item.html?id=140

Current Opinion in Chemical Biology, Volume 12, Issue 1, February 2008, Pages 72-77

What’s the problem?

Confronting the Biomarker Verification Bottleneck

New FDA-Approved (CLIA) Diagnostic Protein Tests in Serum/Plasma Declined for the Last Decade

Anderson, N. L. Anderson, N. G., The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics (2002) 1:845-67.

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Successful Clinical Biomarkers Found So Far by Proteomics

L Anderson, Plasma Proteome Institute

1. Inefficient biomarker discovery processes

2. Lack of verification technology & pipelineSourcing new markers

3. No solid theory or uniform framework of biomarker discovery

4. Slow adoption of new technology platforms

5. Combining patented markersMultiplex panels

6. Lack of regulatory precedents (FDA)

7. Slow physician acceptance

8. Poor public understanding of IVDHealthcare system

9. Positioning of the IVD industry in healthcare

. Major Barriers to New Protein IVD Tests

ChallengesTissues versus fluids

Validation:

In vitroIn vitro In vivoIn vivo

Identify Lung-induced Endothelial Cell Surface Proteins

2D gels analysis

MS/MS identify target proteinsSPECT imaging after injection of APP antibody

Phil Oh, et al. NATURE 2004

Proteomics analysis + SPECT analysis (Animal)

Validation in vivo: Medical Imaging

Payload probeLabelTracerTherapeutic

Targeting LigandAntibodyLow-weight organic

moleculepharma chemistryViruses (gene therapy)

Molecular Target

Targeted Probes Medical ImagingHigh-sensitivity, high-resolution in vivo imaging

In Vivo Molecular Imaging

Drug Development - Bring to market life saving drugs tailored to individual’s genetic make-up faster

Therapy Monitoring - See drugs hitting in-vivo targets in real time

Functional genomics/molecular diagnosis

Molecular and functionalimaging for disease diagnosis and targeted therapeutic

Iterative Paradigm Between Top Down Imaging and Bottom Up Biology

ConclusionsBioinformatics algorithms play a key role in protein biomarker discovery− Challenges in improving the reliability, consistency,

and robustness of algorithms

The gap between biomarker discovery and biomarker validation is a major barrier to application of biomarkers in clinics.− The validation of protein biomarker should be

changed from in vitro to in vivo

Improving diagnostic medicine involves major scientific, technical and policy challenges

Thank You