Monday, September 26th

7:30-8:30 Workshop Registration and Morning Coffee

8:30-8:35 Welcoming Remarks 
Julia Boguslavsky, Conference Director, Cambridge Healthtech Institute

Sample Enrichment and Multi-Dimensional Separations

8:35-8:40 Chairperson's Opening Remarks

8:40-9:05 Developing Ion Mobility Spectrometry for Plasma Proteomics 
Dr. David E. Clemmer, Robert and Marjorie Mann Chair; Chairman, Department of Chemistry, Indiana University
A significant complication associated with the analysis of complex mixtures that are encountered in proteomics arises because of sample complexity. This issue is complicated in the analysis of plasma by the fact that proteins may exist at vastly different concentrations. This talk will focus on the development of ion mobility techniques for the analysis of the human plasma proteome. Because mobilities of ions through gasses are much greater than in condensed phases it is possible to extend the complexity of samples that can be handled by mass spectrometry. The approach also has advantages for extending the dynamic range of measurements. A preliminary map of the human plasma proteome will be presented.

9:05-9:30 The Development of a Platform for Serum Proteomics Using Multiple Lectins (M-LAC): Characterization of Glycoprotein Biomarkers in Breast Cancer Patients 
Dr. William S. Hancock, Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University
In this study, we used multi-lectin affinity chromatograph (M-LAC) to isolate the glycoprotein fraction of serum samples collected from controls and breast cancer patients. After trypsin digestion of the enriched glycoprotein fraction, the peptides were analyzed by nano-LC coupled to a linear ion trap mass spectrometer, and the protein biomarkers were identified from the assigned peptides. From the total of 10 samples, 1400 proteins were characterized, including the identification of some known breast cancer markers, such as Her2, BRCA2, CEA and P53. The level of these biomarkers was obtained by peak area measurement of selected diagnostic peptides. We also performed pathway analysis using the gene ontology parameters (Funcassociates) and found changes in a number of pathways that can provide insights into the disease.

9:30-9:55 Multi-Dimensional Differential Proteomic Analysis Platform for Biomarker Discovery in Brain Injury
Prof. Andrew Ottens, Associate Director, Neuroscience, Center for Neuroproteomics and Biomarkers Research, McKnight Brain Institute, University of Florida
Presented is a novel platform developed to selectively characterize differential protein changes as putative biological markers of stroke and traumatic brain injury. Two-dimensional bi-phasic ion exchange ­ gel electrophoresis (CAX-PAGE) differential protein targeting is followed with reverse phase chromatography tandem mass spectrometry (RPLC-MSMS) for two-dimensional peptide differential validation and protein identification analysis. First dimensional retention is enhanced to 88%, with 94% second dimensional reproducibility, while 89% of differential findings are confirmed by peptide analysis in 85% of cases which distinct proteins are identified. CAX-PAGE compared with standard 2D-DIGE permits differential analysis without expensive fluorescence dyes, solubilizes hydrophobic proteins, extends mass range to 300 kDa and maintains high resolving power at a peak capacity up to 7600. The platform provides rapid proteome screening, effectively reducing putative-targets to a manageable number for validation by immunological assays.

9:55-10:30 Networking Coffee Break

10:30-10:55 Intact-Protein-Based High-Resolution Three-Dimensional Quantitative Protein Scans for Biomarker Discovery
Dr. Hong Wang, Director Mass Spectrometry Facility, Public Health Sciences, Fred Hutchinson Cancer Research Center

10:55-11:20 Synergy of Multiple Protein Separation Technologies Prior to MS Expands the Observable Proteome While Allowing Quantification and Detection of Post-Translational Modifications and Isoforms
Dr. Jennifer Van Eyk, Director of The Johns Hopkins NHLBI Proteomics Center; Director of the Bayview Proteomics Group; and Associate Professor of Medicine, Division of Cardiology, Biological Chemistry and Biomedical Engineering, Johns Hopkins University
Robust biomarkers of evolving disease processes most likely will consist of proteins that are cell, organ and/or disease stage (through disease-induced modifications (PTM)) specific. Hence, the proteomic discovery process must quantitatively monitor protein changes in tissue or serum with obtaining information about the protein isoform and PTM status. We can maximize proteome (or subproteome) by combining protein separation by 2-dimensional gel electrophoresis and 2- dimensional liquid chromatography, as these methods detect only 10-20% overlap in proteins, while assessing all three intrinsic properties of proteins which allows characterization of isoform and disease induced PTM.

11:20-11:45 A 4-D Protein/Peptide Proteomics Analysis Strategy Enables Detection of Plasma Proteins Over a Nine Orders-of-Magnitude Abundance Range
Dr. David W. Speicher, Professor & Chair, Systems Biology Division; Director, Proteomics Laboratory, The Wistar Institute
We recently developed a powerful plasma protein profiling method utilizing three orthogonal protein separations (immunoaffinity depletion, microscale solution IEF, and 1-D SDS PAGE), followed by pixelation of the 1-D gels and LC-MS/MS analysis on a linear ion trap mass spectrometer. When human serum was analyzed, more than 2,700 proteins spanning more than nine-orders-of-magnitude were identified using HUPO criteria for high confidence assignments. A substantial number of proteins known to be present in human serum in the 1-100 ng/ml range and a few proteins in the pg/ml range could be identified using this method. This method is a powerful tool for discovery of serological biomarkers of cancers and other human diseases as it can detect a substantial number of low abundance proteins that are present in serum or plasma at concentrations less than 100 ng/ml.

Luncheon Technology Solutions Showcase

12:00-12:30 New Methods for Biomarker Discovery Using Accurate Mass LC-MS for Endogenous Metabolites and Proteins Mr. Keith Compson, Life Science Business Manager, Waters Corporation   Two LC-MS based methodologies that allow the determination of relative changes in abundance of proteins and metabolites from complex mixtures will be presented. Both use statistical approaches that rely on reproducible chromatographic separations and mass accuracy of OA-TOF mass spectrometers. We have determined changes in relative abundance of ions representing both metabolite and protein concentrations in clinical samples from diseased patients and controls.

12:30-12:45 High Efficiency, High Accuracy Protein Characterization for Untargeted and Targeted Proteomics Studies
Dr. Jean-Pierre Wery, Chief Scientific Officer, Indiana Centers for Applied Protein Science
INCAPS will illustrate the performance of its technologies by describing the results of two studies. The first study analyzes proteomics differences between two cancer cell lines (resistant and sensitive). The second study characterizes the phosphorylation sites of a protein kinase in its active and inactive state.

12:45- 1:00 Luncheon Technology Solutions Showcase		Sponsored by
	Advanced Workflows for Protein Biomarker Discovery, Identification, and Validation Dr. Dominic Gostick, Directot, Proteomics TOF MS Product lines, Proteomics and Small Molecule Division, Applied Biosystems This presentation will introduce innovative LC/MS workflows and systems for identification and quantitation of protein biomarkers. Integrating LC/MS technology with tagging chemistry and application specific software enables simultaneous, high confidence identification and quantitation of putative biomarkers. This talk will show examples of these technologies applied to identifying putative biomarkers in brain tumor tissue, as well as a targeted, high sensitivity, early validation workflows in serum.

1:00- 1:15 Strategies for the Discovery of Protein and Peptide Biomarkers in Human Plasma Dr. Jennifer Sutton, Project Leader, High-performance FT-MS, Thermo Electon This presentation will cover the various aspects of HT sample processing and analysis for the discovery of protein and peptide markers in human plasma. An overview will be provided of the general workflows currently implemented at Thermo BRIMS Center to deplete and fractionate plasma prior to top-down, and bottom-up MS analysis. An emphasis will be placed on label-free methodologies for robust, statistically significant discovery of up/down-regulated peptides using a model of myocardial infarction, as well as on cutting-edge techniques for the thorough top-down characterization of native proteins using FT MS.

Sponsored by

1:15- 1:30 Technology Short Talks (Sponsorship Available- Contact Arnold Wolfson at 781-972-5431 or awolfson@healthtech.com)

Top-Down vs. Bottom-Up Approaches to Protein Biomarker Discovery

1:45-1:50 Chairperson's Opening Remarks

1:50-2:15 Application of a Hybrid Linear Ion Trap/Fourier Transform Mass Spectrometer for Biomarker Discovery
Dr. Ronald C. Hendrickson, Director Proteomics, Molecular Profiling, Merck Research Laboratories

2:15-2:40 Multiplexed Kinase Activity-Based Proteomic Profiling using High-Throughput Mass Spectrometry
Dr. Timothy D. Veenstra, Director, Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc.
Phosphorylation is arguably the most critical event central to signal transduction within the cell. Approaches to measure sites of phosphorylation have historically focused on individual proteins, however, with the advent of improved mass spectrometry (MS) and sample preparation methodologies it is now possible to identify phosphorylated residues at an unprecedented global scale. While identification of sites of phosphorylation enables interesting insights, this technique does not result in a direct measurement of the activity of specific kinases. To this end, we have developed an approach that takes advantage of the high mass accuracy and dynamic range of Fourier transform ion-cyclotron resonance MS to measure proteome-wide changes in kinase activity. This methodology has implications for measuring changes in kinase activity on a global scale and can be customized to specifically measure changes in specific signal transduction pathways and is extendable to other types of enzyme activities. 

2:40-3:05 Emerging Work Flows for Precision Proteomics Using Tandem Mass Spectrometry Above 5 kDa: Separations, MS Engines, and Associated Informatics
Dr. Neil L. Kelleher, Assistant Professor of Chemistry, Department of Chemistry, University of Illinois
The emerging "Top Down" approach to protein and proteome analysis will be described. It combines the tailored separations with a Quadrupole/Fourier-Transform hybrid mass spectrometer (Q-FTMS) and custom software to enable efficient characterization of biological events that change the mass of protein molecules from that predicted by an annotated genome sequence. Top Down Mass Spectrometry holds promise for improving diagnostics and allowing a deeper understanding of the signaling and regulatory "codes" eukaryotic cells use that are written in the language of post-translational modification. Protein examples from Archaea, yeast, and human nuclei will be described, including characterization of histone modifications using a new database strategy termed "shotgun annotation". Top- and even "Middle-Down" strategies of protein analysis are of growing interest. New products are working together to establish new work flows wherein tandem mass spectrometry above 5 kDa provides highly valuable information not readily obtained using existing proteomic methods. 

3:05-3:55 Exhibit Hall Opening, Refreshment Break with Poster and Exhibit Viewing

3:55-4:20 Top-Down and Bottom-Up Proteomics: From Identification to Validation
Dr. Jim Stephenson, Senior Program Director, Mass Spectrometry Research, Research Triangle Institute
This presentation will focus on the strengths and limitations of bottom-up and top-down proteomics. Particular emphasis will be placed on filtering data for false positive and negative identifications, the role of orthogonal separation technology, and gene finding approaches for biomarker identification. A review of relevant instrumentation in the field and its application to protein and peptide analysis is also included. Emerging topics covering post-translational modifications and mutation analysis and their role in biomarker identification is discussed.

4:20-4:45 Mass Spectrometric Top-Down Proteomics Complements Bottom-Up Proteomics
Dr. Maria Esteban Warren, Assistant Director, UNC-Duke Proteomics Core Facility, UNC School of Medicine
While mass spectrometric bottom-up proteomics-the analysis of peptides-is distinguished by its great capability of handling complex mixtures and its high sensitivity, allowing the analysis of peptides present at attomoles, top-down proteomics-the analysis of intact proteins or lager protein fragments-might provide information about the modification stoichiometry and status in proteins. Recent improvements in technology, particularly in the Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometry (MS), permits the detection of isotopically resolved proteins and accurate molecular weight (MW) determination within an error rate of a few parts-per-million, even for high-molecular weight proteins. In addition, the accessibility to different dissociation mechanism in FTICR-MS allows the sequencing of intact proteins (up to a MW of 45 kDa) direct in the mass spectrometer. We have used top-down approaches to characterize the stem-loop binding protein (SLBP) from Drosphilia and have identified a several modifications including a stoichiometric phosphorylation site that is crucial for protein function. Making use of the higher sensitivity of bottom-up approaches, we have shown that this site is highly conserved in different species, thus this study demonstrates the complementary of top-down and bottom-up proteomics.

5:15-6:30 Reception in the Exhibit Hall

For more information please contact: 
Julia Boguslavsky, Conference Director, Cambridge Healthtech Institute
Phone: 781-972-5482 or E-mail: juliab@healthtech.com

For sponsorship information please contact:
Arnie Wolfson at 781-972-5431 or awolfson@healthtech.com.