Thursday, September 9, 2004

PROTEIN BIOMARKER DISCOVERY AND VALIDATION: 
Proteome Profiling of Plasma/Serum and Other Tissues

8:30-8:35    Chairperson's Opening Remarks
Dr. O. John Semmes, Director, Center for Biomedical Proteomics; and Associate Professor, Dept. of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School

8:35-9:05    Applying Mass Spectrometry to the Hunt for Cancer Biomarkers 
Dr. O. John Semmes 
Our research involves the development and application of novel proteomic approaches to the discovery of disease biomarkers. We employ a variety of mass spectrometry techniques including LC-MS/MS, MALDI-TOF/TOF and SELDI-TOF in the identification and analytical detection of cancer biomarkers for clinical diagnostics and prognostics. Discussed will be biomarkers for the detection of prostate, nasopharyngeal and hematopoietic cancers.

9:05-9:35    Rapid Profiling and Identification of Protein Biomarkers for Pharmaceutical Drug Discovery
Dr. Scott E. Warder, Associate Research Investigator, Proteomics, Abbott Laboratories
The discovery and translation of proteomic profiles or marker panels from the discovery phase to a pharmaceutical drug development assay format is extremely challenging. Our strategy is to use complementary technologies, including multidimensional liquid chromatography, MALDI-MS, and FT-ICR, to discover and identify peptides or proteins in the blood proteome that may be utilized as markers of pharmacodynamics, efficacy or stratification. An integral component of the platform is our ability to rapidly triage and identify the peptides/proteins that are regulated. The identity of the markers potentially provide a mechanistic link to a specific target or disease state, and allow appropriate reagents to be generated for marker validation and assay development. Case studies will be presented to demonstrate many aspects of the process.

9:35-10:05    Biomarker Discovery Using a nanoLC dual-ESI FT-ICR Mass Spectrometry Platform
Mr. Kenneth Johnson, Mayo Proteomics Research Center, Mayo Clinic College of Medicine
Mass spectrometry-based platforms are now widely employed in biomarker discovery. We have developed a platform based on nanoLC-dualESI FT-ICR mass spectrometry which affords reproducible retention times (< 6 seconds), high mass measurement accuracy (< 1 part-per million), and reproducible ion abundance (< 62% CV at the 95% confidence interval of the mean). The first two attributes allow for comparison of molecular signatures across diverse patient populations while the latter allows for the direct comparison of ion abundances without the use of internal standards, a pre-requisite for the analysis of the low molecular weight fraction of serum 
and plasma.

10:05-10:35    Characterization of the Proteome of Biological Fluids for Biomarker Discovery
Dr. John E. Hale, Research Advisor, Integrative Biology Division, Lilly Research Labs 
Application of proteomic technologies to biological fluids such as plasma and cerebrospinal fluid promises to revolutionize biomarker discovery. In order to correctly interpret changes in protein levels in body fluids many sample parameters must be understood. These parameters include biological variability, alterations in protein levels introduced through sample handling and processing and the impact of non-compartmental contamination. We have evaluated several of these parameters and their effect on protein levels biological fluids as assessed by mass spectrometry. Several of these findings will be discussed.

10:35-11:15    Coffee Break with Exhibit and Poster Viewing

11:15-11:45    Antibody Microarrays and Two-Color Rolling Circle Amplification for Probing Cancer- Associated Serum Protein Profiles
Dr. Brian B. Haab, Scientific Investigator, The Van Andel Research Institute
The profiling of proteins in biological samples using antibody microarrays has the potential to yield new discoveries. In a step toward further enabling that capability, we developed the use of two-color rolling-circle amplification (Two-Color RCA) to measure the relative levels of proteins from two serum samples that have been captured on antibody microarrays. We are using this tool to study the associations of many circulating tumor and host response markers with a variety of cancers. The presentation will cover the performance and optimal use of the technology, in addition to the biological significance and potential clinical usefulness of the observed protein alterations in the sera of cancer patients.

11:45-12:15    The Diagnostic Potential of the Human Plasma Proteome 
Dr. Leigh Anderson, Founder & CEO, Plasma Proteome Institute 
The technical challenge presented by the plasma proteome is enormous, exceeding the capabilities of current discovery proteomics technologies by a wide margin. An alternative approach, namely to focus on a limited number of sequence-specified proteins, offers the possibility of increased sensitivity, precision and throughput - characteristics required in the marker validation studies required to progress candidates towards commercial in vitro diagnostic tests. As a first step in assembling a list of candidate markers, we have merged four independent plasma accession lists, three from published multidimensional separation/identification strategies plus an extensive search of the biochemistry literature, into a single non-redundant list of 1,175 distinct gene products observed in plasma. To enable reproducible, high-throughput measurement of low abundance candidates from this and other target lists, we are developing a method denoted SISCAPA for quantitation of peptides in complex digests using immobilized anti-peptide antibodies. These affinity supports enrich specific peptides together with spiked stable-isotope-labeled internal standards of the same sequence, providing internally standardized MS quantitation. SISCAPA appears suitable for rapid generation of assays for defined candidate marker proteins, and should facilitate systematic validation of diagnostic protein panels in large sample sets. 

NEW TECHNOLOGY SHOWCASE II 2:15-2:45     Sponsored by: Novel Biomarkers for Early Diagnosis and treatment monitoring of Alzheimer’s Disease  Mr. Eric Fung, Vice President of Clinical Affairs, Ciphergen Biosystems Inc A comprehensive proteomic investigation of human CSF samples was performed using SELDI-TOF mass spectrometry to discover and characterize protein biomarkers of Alzheimer’s Disease (AD) and related dementias. A total of 237 CSF samples were profiled including 98 AD, 31 Frontotemporal Dementia, 29 Dementia with Lewy Body and 88 normal controls. A panel of over 50 biomarkers was shown to be differentially expressed in AD vs. normal and/or non-AD dementia. All of the markers characterized to date reflect different aspects of AD pathophysiology including plaque formation, neuronal degeneration and inflammation.  2:45-3:00    Advances in the Identification and Quantitation of Protein Biomarkers by Mass Spectrometry Dr. Tony Hunt, Applied Biosystems 3:00-3:15 Peptide Biomarker Discovery Platform for Clinical Proteomics Dr. Peter Schulz-Knappe, CSO, Biovision AG

3:15-3:55    Refreshment Break with Last Chance Exhibit and Poster Viewing

What is an "Ideal" Proteomic Biomarker? 

3:55-4:00    Chairperson's Opening Remarks
Dr. Stephen Naylor, CSBi and Division of Biological Engineering, MIT; and Dept. of Genetics and Genomics, Boston University School of Medicine    

4PROTEOMIC SIGNATURES
4:00-4:30    Use of the Serum Fragmentome for Clinical Diagnostics
Dr. Emanuel F. Petricoin, Co-Director, NCI-FDA Clinical Proteomics Program, Senior Principal Investigator, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration
Recently, we have discovered that almost all of the low molecular weight entities that we have been profiling by mass spectrometry exist in a bound state with circulating carrier proteins. These carrier proteins, like albumin, sever to harvest these fragments and low molecular weight molecules, amplifying their overall signal. We have taken advantage of this finding to sequence and identify over 800 specific fragments in ovarian cancer, many of these with over 2 peptide hits. Moreover, we have validated the differential existence of these molecules by western blot analysis. We propose that this method offers an entirely new approach to biomarker discovery. Low abundance and low molecular weight molecules never before known to exist in the circulation can be identified, and if validated, may be used for clinical applications.

4IDENTIFIED PROTEINS
4:30-5:00    Approaches to the Identification and Development of Protein Biomarkers
Dr. Scott D. Patterson, Director, Medical Sciences, Amgen Inc.
Pharmacodynamic biomarkers inform the drug development process. Broad-based analysis platforms analyzing both transcripts and proteins can be used to identify candidate biomarkers that would be measured as their protein products. Development and validation of specific assays for the measurement of proteins in biological fluids or the post-translational modifications within cell populations can be used to examine the drug exposure-response relationship. Considerations for such approaches will be discussed.

4POST-TRANSLATIONAL MODIFICIATIONS
5:00-5:30    Disease Staging and Risk Stratification Biomarkers: Disease-Induced Protein Modifications
Dr. Jennifer Van Eyk, Associate Professor, Medicine, Bayview Proteomic Center, Johns Hopkins Univ.
During the development of disease, proteins undergo specific and selective disease-induced post-translational modifications. These changes directly reflect the disease process giving rise to a unique set of biomarkers with specificity for either the stage or severity of the disease. The identification and characterization of the protein modifications either within the diseased organ or in one or more body fluid, is especially powerful in acute disease when there is insufficient time for large scale genomic changes. For example, during acute myocardial infarction the cardiac specific isoform of troponin I, troponin T and myosin light chain 1 (three key contractile proteins located specifically within the heart) are selectively proteolyzed and/or phosphorylated with increasing duration and severity of ischemia. With loss of membrane integrity, the intact and any modified forms that are present in the tissue are released into the blood. In a similar manner, the skeletal muscle isoforms of these proteins can be detected in their ischemic induced modified forms with increasing respiratory fatigue and injury. In both acute injury settings, detection of one or more disease induced modified forms correlates to increased risk and poor outcome.