8:20 Molecular Targets & Diagnostics: Critical Role of Sample
Preparation in Discovery James
L. Wittliff, Ph. D., M. D. hc, FACB, Professor of Biochemistry & Molecular
Biology, James Graham Brown Cancer Center, University of Louisville
Clinically relevant genomic and proteomic test development using human tissue
specimens requires specialized collection, handling and cryopreservation
methods for generating reliable analyses. Although global gene expression
assays of intact cancer biopsies are utilized to distinguish patterns,
validation of mRNA expression of specific gene sets by techniques such as
quantitative PCR is essential using well characterized samples. Non-distructive
procurement of pure cell populations from frozen and formalin-fixed,
paraffin-embedded tissues by Laser Capture Microdissection and optimized
methods for RNA and protein analyses enhance identification of candidate
molecular targets for development of drugs and diagnostics. These approaches
must be complemented by well annotated records of patient characteristics,
tissue pathology and clinical outcome
9:00 Opportunities and Obstacles in Proteomic Analysis of Biofluids Sunny Tam, Ph.D., Research Associate Professor, University of Massachusetts
Medical School
Proteomic analyses of biofluids, such as plasma or amniotic
fluid, traditionally have the same obstacles due to the presence of abundant
proteins and large dynamic range of protein concentration. Recent fractionation
reagents have helped in the isolation of abundant proteins to allow the
examination of lower abundant proteins. We have examined plasma from a diabetic
rat model and important clinical diseases after adequatefractionation schemes.
The proteomic finding from gel based and iTRAQ based studies have yielded
interesting observations after bioinformatics analysis. Furthermore, the
biological significances of the differential protein expression have been
validated with traditional biochemical methods.
9:40 Grand Opening Coffee Break in the Exhibit Hall
Optimization
10:25 Chairperson’s Remarks
10:30 Protein Quantification and Separation before 2D Electrophoresis
Using Label Free Intrinsic Imaging
Judit Nagy, Ph.D., Proteomics Facility Director, Institute of Biomedical
Engineering, Imperial College London One of the most commonly used protein separation techniques
in proteomics is two dimensional gel electrophoresis (2DE). Although 2DE is
cheap to set up and gives a visual profile of complex protein mixtures, it has
many drawbacks. It is not quantitative, it can be laborious, it cannot separate
the complete protein set and the reproducibility is low. All these factors
result in increased sample replicates needed in the discovery of biologically
relevant protein changes. To avoid running low quality samples on 2D gels and
wasting valuable materials and time, samples are first analysed and quality
checked using deltaDOT’s Peregrine system.
Protein detection is based on Label Free Intrinsic Imaging (LFII™)
developed by deltaDOT. This work-flow not only allows to speed up and improve
the 2DE gel outputs but also gives the opportunity to verify and quantitate
proteins which are identified from the 2D gels by mass spectrometry.
11:00 New Approches of Sample Preparation and Fractionation
Hongshan Li, Ph.D., Senior Principal Scientist, Proteomics, Pall This talk will discuss the advantage and dis-advantage of new
resins (HEA,PPA and MEP) combination of IEX, IMAC and SDR.
11:30 Novel Inactivation Technology that Preserves the in vivo Proteome Mats Borén, Ph.D., Head of Research, Denator Biotechnology AB The talk focuses on the massive ex vivo degradation of the
proteome that usually is provoked in most pre-analytical proteomics workflows.
Rapid inactivation and stabilization of the proteome is shown to be crucial for
analysis of an in-vivo-like proteome instead of the degradome. A novel
methodology utilizing rapid and irreversible heat fixation of the proteome is
presented as a solution to the ex-vivo degradation problem. The benefit of this
new technology is exemplified within several standard proteomic workflows.
12:00 pm Lunch and Learn Workshops (Sponsorship Available) or Lunch on
Your Own
Biomarkers
1:25 Chairperson’s Remarks
1:30 In Situ Synthesis of Protein Arrays Mingyue He, Ph.D, Technology Research, The Babraham Institute In situ protein synthesis technologies exploit cell-free
expression systems to produce protein arrays on glass slides directly from
co-distributed or pre-arrayed DNA. These methods avoid the laborious and costly
processes of DNA cloning, expression and purification of individual proteins and
eliminate the need for storage of functional proteins on the surface over time.
We have developed novel cell-free protein array methods, which not only allow
conversion of arrayed DNA into protein arrays in hours but also ‘print’
multiple copies of a protein array through repeated use of a single DNA array.
Their application for screening proteins will be discussed.
2:00 Integrative Biomarker Discovery Strategies
Martin Latterich, Ph.D., Associate Professor, Pharmacy, University of
Montreal The burgeoning field of personalized medicine will require
the identification and validation of new biomarkers that have prognostic or
diagnostic value. Some of the challenges facing us today are well-defined
patient cohorts, and highly reproducible analytical methods, as well as cost
effective methods of validating often large sets of biomarker candidates. We
have developed an integrated strategy for both the discovery, as well as the
validation of biomarkers from different types of patient samples that includes
aspects of sampling, storage, isobaric tagging, and subsequent MIDAS-MRM
validation.
2:30 Escorting Protein Biomarkers Down the Discovery Pipeline Using Mass
Spectrometry Immunoassay: A Case Study of Work in Progress with Type 2
Diabetes Biomarkers
Chad Borges, M.D., Assistant Research Scientist, The Biodesign Institute,
Arizona State University A protein biomarker of disease is not just "a
protein"—it is something about a protein—generally either a change in
concentration, amino acid sequence, and/or qualitative/quantitative changes in
posttranslational modification. Since human knowledge of protein biochemistry
is far from complete, our laboratory operates on the hypothesis that every
protein is a potential biomarker platform and that all three of the above
biomarker qualifiers must be considered for all proteins. Toward this end, the
application of mass spectrometric immunoassay (MSIA) towards plasma samples
from over 100 individuals has recently facilitated our detection and
preliminary validation of several characterized biomarkers towards Type 2
Diabetes (T2D) diagnosis and monitoring—including a situation in which two
biomarkers are carried by a single protein and monitored by a single mass
spectrometry based assay. A clinical study designed to generate plasma samples
for T2D biomarker validation has been arranged and further MSIA-based high
throughput validation of these biomarkers will continue as soon as these
samples are collected. The nature of these protein biomarkers (and others like
them) strongly suggests the need for mass spectrometry as a diagnostic tool in
the clinical setting.
3:00 Networking Refreshment Break, Poster and Exhibit Viewing
Sponsored by
Protein Analysis
3:45 Microfluidic Cassette for Rapid
Isolation of Granulocytes from Whole Blood for Genomic and Proteomic
Applications Dr Ken Kotz, R&D Operations, Surgery, Massachusetts General
Hospital
4:15 Cerebrospinal Fluid and Proteomic Analyses Pawel Ciborowski, Ph.D., Assistant Professor, Pharmacology and Experimental
Neuroscience, University of Nebraska Medical Center
Proteomic analysis of cerebrospinal fluid (CSF):
CSF sample handling and preparation for various proteomic analyses
Issues related to CSF sample pooling when limited material is available
CSF sample normalization
Validation of proteomic results in CSF analyses
Data interpretation in CSF proteomic analyses
4:45 Development of Protein Chemistry Methods for the Detection of
Protein Post-Translational Modifications (PTM’s) Zhaohui Sunny Zhou, Ph.D., Faculty Fellow, The Barnett Institute of Chemical
and Biological Analysis; Associate Professor, Department of Chemistry and
Chemical Biology, Northeastern University Over one hundred different protein post-translational
modifications (PTM’s) have been reported, playing critical roles in myriad
biological processes in humans. Due to the relative low abundance of these
modified proteins, detection of these PTM’s remains a major challenge in
proteomic research.
Our laboratory has successfully developed several biochemical
and chemical methods to selectively derivatize the modified proteins with
various chemical tags, allowing specific fluorescent staining for quantification
and global profiling, for example. Moreover, by introducing affinity tags,
specific modified proteins can be markedly affinity enriched, drastically
simplifying sample complexity and subsequent proteomic analysis. Examples
demonstrating our approach include protein deamidation, methylation and
homocysteinylation, which play key roles in aging, cancer and cardiovascular
diseases.
