GENOMIC AND PROTEOMIC SAMPLE PREPARATION

Advisors and Chairs
Dr. Thomas Laurell, University of Lund
Dr. Bruce Roe, University of Oklahoma
Dr. Terrence Ryan, Celera Genomics
Dr. David W. Speicher, The Wistar Institute
Dr. James L. Wittliff, University of Louisville

Preconference Short Course Tutorial: Microfluidics for Lab-on-a-Chip
Dr. Helene Andersson, Life Science Manager, Microfluidics and Biotechnology, Silex Microsystems, AB
Dr. Albert van den Berg, University of Twente and MESA+ Research Institute

Standards, Collection, Storage, and Stability
Dr. James L. Wittliff, University of Louisville
Dr. Jean Gabert, CHU, Nord
Dr. Francine Benes, The Harvard Brain Tissue Resource Center
Dr. Rembert Pieper, Large Scale Biology Corporation
Dr. Michael J. Pellini, Genomics Collaborative, Inc.
Dr. James C. Robbins, Whatman International Ltd.

Genomic Sample Preparation
Dr. Bruce Roe, University of Oklahoma
Dr. Duncan Whitney, Exact Sciences Corp.
Dr. Martin Smith, Whatman, Inc.
Dr. Rajiv Raja, Head of Life Science, Arcturus, Inc.
Dr. Pascal Bonaventure, Johnson & Johnson Pharmaceutical Research & Development, LLC
Dr. Yuhai Tu, IBM T.J. Watson Research Center

Proteomic Sample Preparation
Dr. David W. Speicher, The Wistar Institute
Dr. Walid Qoronfleh, Perbio Science AB
Dr. Oleg Chertov, National Cancer Institute
Dr. William Kopaciewicz, Millipore
Dr. Matthew Kuruc, LigoChem, Inc.
Dr. Dmitriy A. Vinarov, University of Wisconsin–Madison
Dr. David B. Krizman, Expression Pathology, Inc.

Preconference Short Course Tutorial

3:00-4:00pm Tutorial Registration

4:00-8:00pm Microfluidics for Lab-on-a-Chip
Dr. Helene Andersson, Life Science Manager, Microfluidics and Biotechnology, Silex Microsystems, ABDr. Albert van den Berg, University of Twente and MESA+ Research Institute
The use of microdevices made by advanced micromachining technologies makes it possible to handle very small quantities or very low flow rates of liquids and gases. This provides new opportunities in the medical instrumentation area, in the pharmaceutical industry, and in many other fields. By the end of the course, the attendees will know the present state of the art in Micro Fluidic Systems (MFS) and have an overview of the application areas. The course will also point out future developments of devices and applications. Special attention will be paid to the most important area of application, that of bioanalytics or "Lab-on-a-Chip." Different approaches and examples of solutions for real-life problems will be discussed.

All those who register for Microfluidics for Lab-on-a-Chip tutorial are cordially invited to attend the Closing Plenary Session of Macroresults for Microarrays on Wednesday, May 14, at 1:30. Please visit: http://www.healthtech.com/2003/mar/index.asp for details about the plenary session.

8:30 Chairperson’s Opening Remarks
Dr. James L. Wittliff, Professor of Biochemistry & Molecular Biology and Research Professor of Surgery, Hormone Receptor Laboratory, University of Louisville

8:40 Development of Standards for Cellular Gene Expression
Dr. Jean Gabert, Professor of Biochemistry and Molecular Biology, Faculty of Medicine, CHU, Nord
Biological standards or reference materials are crucial for validation and standardization of biological assays. For example, lyophilized human plasma containing live viruses have been developed as WHO international standards for nucleic acid amplification techniques (NAT) for the detection of viral pathogens and are distributed by the National Institute for Biological Standards and Control (NIBSC), UK (a WHO collaborating laboratory). Lyophilized K562 cells have been prepared as a potential International Standard for NAT for cellular gene expression analyses in oncology. Data from an ongoing international study, which includes 25 laboratories using real-time quantitative PCR analysis for BCR-ABL RNA quantification, will be presented. Assessment of this material for gene profiling analysis is in progress.

9:10 Quality Control for Gene Expression Profiling in Human Postmortem Brain Tissue
Dr. Francine Benes, Director, Structural Neuroscience Laboratory, and Director, The Harvard Brain Tissue Resource Center
The procedures that we follow to evaluate the quality of RNA extracts and the replicability of the gene expression profiling results will be described. In addition to assessing the ratio between the 18S and 28S RNA peaks using an Agilent Bio-Analyzer, each case in a given cohort is hybridized to its own microarray, but the extracts for a given study are all hybridized to microarrays from a single lot purchased at the same time. The following steps are used to evaluate each scanned chip: (a) visual inspection for artifacts or excessively high background that could distort the readings, (b) examination of the 3’/5’ ratio to ensure that the RNA was of satisfactory quality, (c) assessing the signal for constitutively expressed housekeeping genes, such as actin and G6PDH, and (d) determining the "present" calls to ensure that they fall between 35 and 45%. When all the criteria are met, the data are analyzed using the Affymetrix Data Mining Tool and D-Chip under both low- and high-stringency conditions. Only those genes detected by both programs under high stringency conditions are considered to be meaningful signals.

9:40 Selective Enrichment of Lower Abundance Proteins in Body Fluids
Dr. Rembert Pieper, Director, Protein Chemistry, Large Scale Biology Corporation
A technology has been developed for selective enrichment of lower abundance proteins in body fluids, particularly plasma and serum. It is based on multicomponent immunoaffinity subtraction chromatography and allows high throughput and reproducible sample preparation in projects aimed at the discovery of disease markers via quantitative proteomics. The utility of this technology will be described in proteomic studies on body fluid samples from cancer patients.

10:45 Stable Storage of Proteins at Room Temperature
Dr. James C. Robbins, Technical Customer Service Manager, Genomics/Proteomics,Whatman International Ltd.
Stable storage of functionally active proteins is challenging, often requiring storage at -20°C or use of lyophilized preparations. In the present study we report the development and utility of Protein Saver, a novel storage medium to which protein samples can be applied, allowed to dry, and then stably stored at room temperature for several months. We report data on the stable storage of human plasma prior to 2-D gel electrophoresis, the digestion of gels spots using stably stored trypsin, and the storage of tryptic digests prior to MALDI-MS. Stored proteins can be readily eluted from Protein Saver without the use of specialized elution systems.

11:15 Optimization of Tissue Handling and Extraction to Procure Distinct Cell Types by LCM for Genomic and Proteomic Analyses
Dr. James L. Wittliff
Human tissue collection, handling, and analyses present specific problems for developing clinically reliable genomic and proteomic tests, unlike studies with human tissues or cell lines grown in tissue culture. Laser Capture Microdissection (LCM) represents a major advancement in nondestructive cell sampling technology that can be applied to genomic and proteomic studies. To evaluate differences between normal and diseased cells, LCM is used to isolate structures or distinct cell types and extract them independently for DNA, RNA, or protein analyses under stringent conditions (e.g., RNase-free). Proper tissue procurement, specimen handling, and cryopreservation are essential for the collection of quality information for genomic and proteomic analyses. Following protocols to be presented, gene expression profiles (molecular signatures) have been deciphered for human breast cancers exhibiting various tumor marker phenotypes.

11:45 Transitioning towards Using Large-Scale Association Studies Using Genomics Collaborative’s Global Repository
Dr. Michael J. Pellini, Chief Executive Officer, Genomics Collaborative, Inc.
Evidence is accumulating that the pharmaceutical industry is transitioning towards using large-scale association studies. These studies employ large numbers of well-characterized DNA samples to expedite drug discovery and to improve diagnosis, prediction, prevention, and treatment of disease. Such studies may enable researchers to make critical "go–no go" decisions much earlier in drug development, thereby eliminating the significant costs and substantial loss of time associated with products that previously failed later in development. Genomics Collaborative’s Global Repository® has been designed to enable this transition, and the company has the experience and expertise in handling and using samples collected from more than 120,000 patients for such studies.

1:45 Chairperson’s Remarks
Dr. Bruce Roe, Research Professor of Chemistry and Biochemistry, University of Oklahoma

1:50 Sample Preparation for Genomic Sequencing and Conceptual Planning and Implementation of Automated Processes for High Throughput
Dr. Bruce Roe
As the Human Genome Project reaches its conclusion, in this talk I will look back over the past decade and discuss the growth of automation from its beginnings with automated pipettes to its present-day, high-throughput, 384-well, robotic-based pipetting and sample handling. Since our Genome Center was one of the groups that sequenced approximately one-third of the q-arm of human chromosome 22, the first human chromosome completed, analysis of the genomic features of this representative human chromosome will be presented in light of similar, more recent results from the entire human genome. The sequence of human chromosome 22 was derived from over 20 individuals; the overwhelming similarities (99.8%) and differences (0.2%) between them will be discussed. More recently, we have concentrated our sequencing on orthologous (syntenic) regions of human chromosome 22 from mouse chromosomes 6, 16, and 10 in an effort to further confirm and annotate the ~60% of the predicted genes that have unknown functions and to investigate the evolutionary chromosomal breakpoints present there. In addition, results of sequencing orthologous regions of chimpanzee and baboon, which are 98% and 92% identical to the human genome, will also be presented, and their similarities and differences will be discussed.

2:20 Development of PreGen™–Pure for Purification of DNA from Stool
Dr. Duncan Whitney, Director, Process Engineering, Exact Sciences Corp.
Detection of clinically relevant colorectal-cancer-associated DNA markers in stool has notable technical difficulties. One key to success is a robust and cost-effective means of reproducibly purifying high-quality human DNA from an asymptomatic average-risk patient population. We addressed this challenge by developing an electrophoretic, gel-based affinity capture technology (PreGen™–Pure) that recovers and purifies human DNA from stool. This sequence-specific capture methodology can accommodate sample volumes ranging from 10 ul to 10,000 ul. Data will be presented demonstrating that this methodology is both efficient and highly specific within a complex nucleic acid environment such as with stool samples.

2:50 Novel Filtration Techniques and Devices for the Rapid Isolation and Analysis of Genomic DNA from Biological Samples
Dr. Martin Smith, Director, Technical Marketing, Whatman, Inc.
At present there are no rapid techniques (<20 minutes) available for the high recovery rate isolation of genomic DNA from large volumes of whole blood that can address this market need. We have recently patented the utility of filtration technology to isolate genomic DNA-bearing cells (leukocytes) from whole blood and instantly release genomic DNA from them. The technique exploits blood transfusion leukoreduction technology where leukocytes are efficiently and specifically removed by filtration from red cells prior to transfusion. Our system is totally scaleable and suitable for 10ml of whole blood, is rapid, and produces DNA suitable for all downstream analyses such as PCR, Invader Assay, and real-time PCR. Because the system is scaleable we have also designed a high-throughput microplate format capable of isolating genomic DNA from 96 samples of up to 400 ul of whole blood simultaneously using standard liquid handler devices.

4:00 MicroGenomics: A Novel Platform for Microarray Analysis of Ten Cells
Dr. Rajiv Raja, Head of Life Science, Arcturus, Inc.
We have developed novel tools for microarray analysis of small samples such as laser capture microdissected (LCM) cells and fine needle aspirates that attain improved sensitivity at three stages: (1) capturing pure cell populations through LCM while maintaining integrity of cellular RNA, (2) efficiently recovering good-quality RNA from very small samples through an optimized RNA isolation system, and (3) linearly amplifying messages to provide adequate amounts of amplified antisense RNA for microarrays. When the whole platform is used, considerably higher sensitivity is attained in quantifying differential expression and revealing signatures not seen when whole tissues are assayed. Gene expression studies can be performed using as little as100 picograms of total RNA, or 10 cells. We will present data on various biological applications of this platform, including breast cancer, neuroscience, and developmental biology.

4:30 Gene Expression Profiling of Selected Cells within a Complex Tissue
Dr. Pascal Bonaventure, Scientist, Drug Discovery, Neuroscience, Johnson & Johnson Pharmaceutical Research & Development, LLC
Gene expression profiling of the brain is not trivial due to the marked heterogeneity of neural cells. We have developed an integrated technology consisting of laser capture microdissection, T7-based aRNA amplification, and microarray analysis to allow gene expression profiling of selected cells within a complex tissue. The general principles of this approach and its specific applications to study brain function will be discussed.

5:00 Quantitative Noise Analysis for Gene Expression Microarray Experiments
Dr. Yuhai Tu, Scientist, Physical Sciences & Computational Biology Center, IBM T.J. Watson Research Center
We report a detailed noise analysis for oligonucleotide-based microarray experiments involving reverse transcription, generation of labeled cRNA (target) through in vitro transcription, and hybridization of the target to the probe immobilized on the substrate. By designing sets of replicate experiments that bifurcate at different steps of the assay, we are able to separate the noise due to sample preparation and the hybridization processes. We quantitatively characterize the strength of these different sources of noise and their respective dependence on the gene expression level. We find that the sample preparation noise is small, implying that the amplification process during the sample preparation is relatively accurate. The hybridization noise is found to have very strong dependence on the expression level, with different characteristics for the low and high expression values. A method to evaluate the significance of gene expression fold changes based on the noise characteristics is proposed.

8:30 Chairperson’s Remarks
Dr. David W. Speicher, Professor and Director, Proteomics Laboratory, The Wistar Institute

8:35 A Sample Preparation Platform Technology for Proteomic Applications
Dr. Walid Qoronfleh, Senior Director, Department of Research and Development, Perbio Science AB
We have developed an affinity chromatography platform for proteomic sample prep applications that uses a selected chromatography medium that is dehydrated to form uniform aggregates. The discs will instantly rehydrate upon addition of the protein sample. Discs can be made in assorted sizes (resin volume 15 µl to 3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns), and different ligands can be attached to the matrix. Data will be presented applying SwellGel® discs to high-throughput proteomic applications such as affinity-tag purification, protein desalting, the removal of abundant proteins from serum, and the direct isolation of phosphorylated peptides from protein digests in compatible buffers for mass spectrometry by MALDI or LC/MS.

9:05 Extraction of Peptides from Blood Serum with Simultaneous Precipitation of Large Proteins by Organic Solvents as a Simple Sample Preparation for Mass Spectrometry Analysis
Dr. Oleg Chertov, Senior Scientist, SAIC-Frederick, Inc; National Cancer Institute
It is reasonably assumed that changes in proteins of blood serum or plasma may reflect changes in the pathological state of the organism. Unfortunately, directly comparing proteins in unfractionated serum from healthy and diseased patients is hampered by the presence of several highly abundant proteins. Serum contains peptides from degradation of proteins in serum and other tissues, and the changes in peptide composition are indicative of changes in the physiology of the organism. Because such peptides could be bound to highly abundant proteins such as albumin, their depletion from serum under native conditions may result in loss of potential biomarker peptides. We have developed a method to "extract" peptides from serum under denaturing conditions using organic solvents while at the same time precipitate large abundant proteins to simplify subsequent mass spectral analysis. This sample preparation method provides a simple way to extract serum peptides, enabling them to be compared and identified using different mass spectrometry approaches.

9:35 Comprehensive Protein Profiling of Complex Proteomes Using Microscale Solution Isoelectric Focusing (IEF) Followed by Multifaceted Downstream Analyses
Dr. David W. Speicher
No current protein analysis platform is capable of quantitatively profiling the majority of the 20,000+ proteins in most mammalian proteomes. Hence, initial prefractionation of complex proteomes into a small number of well-separated fractions using microscale solution IEF can substantially improve capacities of either 2-D PAGE or nongel-based separations. Due to protein diversity and limitations of both gel-based and nongel separation methods, the most comprehensive profiling of complex proteomes can be achieved by integrating multiple downstream analysis techniques including high-resolution 1-D gels for large and insoluble proteins, narrow range 2-D gels, and multidimensional chromatography combined with MS/MS. This multifaceted approach is capable of reliably quantitatively comparing at least 5,000 to 10,000 proteins from complex samples.

10:05 Refreshment Break, Poster and Exhibit Viewing10:35 Glycoprotein Enrichment and Bioactive Proteomics
Dr. Matthew Kuruc, Vice President, Business Development, LigoChem, Inc.
Proteins post-translationally modified by carbohydrates are a very important subclass of proteins that have been implicated in many intractable diseases such as cancer. However, the proteomic investigation of glycoproteins has been severely constrained by two factors: harsh enrichment techniques and inadequate electrophoretic resolution. Two solid-phase techniques combined in series overcome many of these limitations. Elastomeric polyelectrolytes provide up to 8X enrichment from plasma. These glycoproteins are then further subfractionated by ProFACT™, a platform based on ligand-specific macrobeads configured in multiwell arrays. As functional integrity is maintained, bioassay measurements advantageously produce digital fingerprints for comparative analysis. Thus, these separation methods reduce complexity and intrinsically provide a pathway to high confidence glycoprotein targets.

11:05 Developments in Proteomic Sample Preparation
Dr. William Kopaciewicz, Research & Development Director, Millipore
Mass Spectrometry has revolutionized protein identification in electrophoresis gels. However, once the protein spot has been excised, a relatively involved series of manip-ulations are required in order to prepare the sample for analysis. Traditionally, sample processing has been conducted using common laboratory plastic ware and home-made solid phase extraction devices. Although functional, this approach has room for improvement. As such, we will discuss alternative systems for gel spot processing that can increase both speed and sensitivity.

11:35 Protein Production for Structural Investigations Based on the Wheat Germ Cell-Free Expression System
Dr. Dmitriy A. Vinarov, Assistant Scientist and Project Leader, Center for Eukaryotic Structural Genomics, University of Wisconsin–Madison
The latest developments in the cell-free protein expression and proteomic sample preparation for structural investigations will be discussed.

12:05 High-Throughput Molecular Profiling of Archival Tissue
Dr. David B. Krizman, President and Chief Scientific Officer, Expression Pathology, Inc.
Archival formalin fixed, paraffin embedded (FFPE) tissue is a vast source of human clinical material; however, standard processing methodologies limit the use of such tissue in high-throughput protein analysis applications. To overcome this limitation we have developed a simple, efficient methodology for microarray-based analysis of proteins directly from FFPE tissue. This system is based on a proprietary sample preparation method, which enables collection of specific histological features in FFPE tissue and preservation of proteins from that tissue for direct identification and quantitation. The system also employs a noncontact protein spotting technology that enables accurate arraying of precious tissue material. Enhanced fluorescent signal detection methods provide a wider dynamic range of signal and make it possible to quantitate protein levels and perform expression analysis of low- to mid-abundant proteins. Results will be presented to demonstrate that this system enables identification and quantitation of proteins across multiple FFPE tissue samples in a high-throughput array format, in a way that links the protein data directly to corresponding histopathology information about each tissue.

Microfluidics Sample Preparation (Joint Session)

1:45 Chairperson’s Remarks
Dr. Thomas Laurell, Professor, Department of Electrical Measurements, University of Lund

1:50 Microfluidics for Broad Applicability and Broad Acceptance Areas
Dr. Mike McNeely, President and Chief Executive Officer, BioMicro Systems, Inc.
Of many microfluidic technologies, few have the characteristics necessary for broad applicability and broad acceptance. Some relevant characteristics include simple yet sophisticated processing and easy adoption and implementation, a clear improvement over existing technologies, all at an acceptable price. BioMicro’s PFC™ technology has been shown to address multiple technological needs simply and inexpensively. Examples of its application will be shown in microarray processing, diagnostics, and genomics, demonstrating its present and future value in these areas.

2:20 Chip-Integrated Solid-Phase Sample Preparation and Enrichment Prior to MALDI-TOF MS
Dr. Thomas Laurell
Solid-phase microextraction is widely used for proteomic sample preparation in standard robotic sample handling equipment. In hand with the development of lab-on-a-chip concepts the need for new miniaturized sample handling protocols becomes evident. Our developments on chip-integrated solid-phase sample preparation and enrichment prior to MALDI-TOF MS readout will be overviewed. In line with the chip development the importance of proper microfluidics in the chip will also be emphasized.

2:50 Microfluidic HPLC Systems for Proteomics and High Throughput Small Molecule Analysis
Dr. Karen Hahnenberger, Director of Biological Assays, Eksigent Technologies LLC
Microfluidic systems play an important role in proteomics, where the ability to control the separation of peptides at nanoscale flow rates improves the mass spectrometer's ability
to detect low-abundance proteins. Achieving precise flow control at low flow rates has proven difficult and has often required flow splitting techniques. We will describe the
development of a microfluidic HPLC system that provides direct, pulseless control at separation column flow rates of 10 to 500 nL/min, without the need for flow splitting. The
NanoLC's microfluidic flow control system provides the ability to reduce the separation column flow rate rapidly while maintaining the gradient profile. This capability, known
as peak parking, provides additional time to run tandem MS on chosen peptides. System data for peptide separations, sensitivity data and peak parking results will be presented.
We will also describe a microscale HPLC system optimized for 300 mm id columns with flow rates of 0.5 - 10 mL/minute. This system makes use of a microfabricated 45 nL flow
cell for UV detection (200-380 nm). Results for system reproducibility, sensitivity and separation efficiency will be presented.

3:20 Refreshment Break

3:45 Microfluidic Platform for Complex Sample Analysis: Integrated On-Chip Sample Preparation, Cellular, and Chemical Analysis Starting from Raw Samples
Dr. Bernhard H. Weigl, Micronics, Inc.
A microfluidic platform and devices for the analysis of complex samples such as whole blood and biological cell suspensions are presented. These devices use at their core flow structures based on Laminar Fluid Diffusion Interfaces (LFDIs). The method is being applied to, among other examples, the extraction and subsequent detection of drugs and small proteins from whole blood and cell suspension samples, the lysing and detection of blood particles, and the chemical detection of various constituents in whole blood and urine. These devices combine several different individual analysis steps in a single on-chip process that does not require user intervention. The analysis frequently involves sample cleanup, cell lysing, extraction and concentration of analytes, concentration determination, and on-chip waste storage.

4:15 Advanced Technologies for Interfacing Microfluidic Devices to High Throughput Analysis and Screening Instrumentation"
Dr. Manish Deshpande, Vice President of Engineering, Teragenics, Inc.
One of the major impediments to the use of microfluidic devices in drug screening and other high-throughput analytical applications has been the lack of appropriate interfaces between standard robotic lab automation equipment and microfluidic systems. In this presentation, a novel way of interfacing robotic automation systems with microfluidic chips will be presented. The technology can be applied to applications in capillary electrophoresis, protein separation, on-line gradient spotting, compound dispensing, serial dilution and Mass Spectrometry applications. Experimental results obtained in applications such as drop dispensing, direct injection into a microchannel and formation of a sample plug for CE separation will be provided.

4:45 Close of Conference

CALL FOR POSTERS
Cambridge Healthtech Institute encourages attendees to gain further exposure by presenting their work in the poster sessions. Please fill out the registration form, with the poster title and primary author. To ensure inclusion in the conference CD, a one-page summary must be submitted and registration must be paid in full by April 4, 2003. Click here for poster instructions

CONFERENCE VENUE
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HOTEL INFORMATION
Seaport Hotel
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Room Rate: $189 S/D
Cut-off Date: April 21, 2003
Please call the hotel directly to make your room reservation. Identify yourself as a Cambridge Healthtech Institute conference attendee to receive the reduced room rate. Reservations made after the cut-off date or after the group room block has been filled (whichever comes first) will be accepted on a space-and-rate-availability basis. Rooms are limited, so please book early.

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