Confirmed Poster Participants:
 

"Mining the Yeast FLEXgene Repository for New Checkpoint Control Genes"

Bhupinder Bhullar, Harvard Medical School, bhullar@hms.harvard.edu

Determining the function of the large number of unknown proteins in the genome requires the development of high-throughput functional assays.  We are using in vivo high-throughput assays to screen for proteins involved in cell cycle checkpoint function. Checkpoint proteins monitor growth and division events during cell cycle for errors.  In the human, loss of checkpoint proteins (eg. pRb, p53, BRCA1, ATM) predisposes cells to acquire genomic abnormalities at an accelerated rate and ultimately to cancer.  We constructed a high-throughput functional proteomics approach in yeast to screen for proteins whose ectopic expression disrupts normal checkpoint function. To accomplish this, we created clones for the predicted ~6000 Saccharomyces cerevisiae ORFs using the Gateway recombination cloning system (Invitrogen) to construct the YeastFLEX  (Full Length EXpression) Library.  Subsequently, we transferred the cloned ORFs into a yeast expression vector (CEN, URA) under the control of a galactose inducible promoter (GAL1-10) and introduced the expression clones into our yeast tester strains.  All genes used in the screen are confirmed to be full length and wild type by sequence analysis. To identify DNA damage checkpoint lethal genes, we developed a semi-automated high-throughput protocol to screen the YeastFLEX library for genes that cause cell death in the presence of damaged DNA.  So far, we have screened approximately 900 sequence verified genes in our checkpoint assay and have identified three candidate checkpoint disruptor genes, which we are currently characterizing.

"Sample Solubilization Effects Resolution in 2-Dimensional Electrophoresis with IPG "

Marcia Goldfarb, Anatek-EP, anatekep@maine.rr.com

Solubilization of samples for 2D electrophoresis is a key step in optimizing pattern resolution. Several basic recipes were formulated for use with the 1st dimension carrier ampholyte gels. These consisted of urea or CHES, detergent and a disulfide bond reducing agent.  Different biological samples were optimally separated by different recipes.  The recipe for serum and plasma utilized CHES and SDS, and is often heated to 95 0C for 5 minutes.  Cellular fractions use a variation of urea, NP-40 (now IGEPAL), DTT or mercaptoethanol.  Work with IPG (immobilized pH gradient) strips indicates solubilization of the sample is still an important step for good resolution and requires  a trial matrix of recipe configurations which include some concentration of ampholytes to set up the optimum protocol. This work looked at solubilization of a human milk sample for separation on 13cm 3-10NL IPG strips (Amersham Bioscience) with four combinations of detergents; CHAPS and IGEPAL, and ampholytes, Pharmalytes 3-10 for IEF and BioRad 3-10.  Sample was entered on cathodic end of gel. Electropherograms reveal a complex relationship between detergent and ampholytes.  No one combination gave superior resolution in all areas of the gel.  BioRad 3-10 ampholytes were an essential ingredient for focusing high molecular weight molecules, greater then 70kDa.  The rest of the gel may be well resolved, while high molecular size proteins appear to not have focused at all with other recipes.  Identification of all proteins in a complex mixture will require use of various solubilization strategies for all to be elucidated.

"Identification of Cardiac Protein Abnormalities in Diabetes Mellitus by LC-MS/MS"

Edward Feener, Joslin Diabetes Center, Edward.feener@joslin.harvard.edu
 

--Abstract Unavailable--

"Resonance Light Scattering on Protein Microarrays: Highly Sensitive Parallel Detection of Cytokines"

Gillian Wotherspoon, Genicon Sciences, gwitherspoon@geniconsciences.com

Extensions to this capability involve relating the results from 2D electrophoresis with related experimental data. This includes data internal to the organization (other protein, gene, tissue or cell databases) and data external to the organization (protein databases generated by third party organizations.) The key objective of this work is to find correlations among 2D gel experimental results with know systems biology behavior.

"Eprogen's Protein Discovery Laboratory Featuring ProteoSep(tm) Technology"

Timothy Barder, Eprogen  tbarder@eprogen.com  

Eprogen, Inc's ProteoSep is a novel protein discovery chemistry and software platform that addresses the need for improved analytical techniques in high-resolution protein anaysis.  It is offered through Eprogen's Protein Discovery Laboratory for those involved in screening potential new drug candidates and evaluating new approaches to disease therapy.

ProteoSep is an all-liquid phase protein mapping technology that uses standard HPLC instrument technology to produce high resolution 1D and 2D maps of complex protein systems and serves as an alternative to 2D PAGE.   

Proteins are separated in the first dimension based on pI information using a unique high performance chromatofocusing (CF) column developed by Eprogen. Subsequent analysis of these pI fractions with Eprogen's proprietary reverse phase NPS® columns provides for the second dimension separation information based on hydrophobicity.  A "2D protein map" is produced using Eprogen's ProteoSep Software Suite, displaying the proteins in bands like that presented in a 2D PAGE gel. 

Due to ProteoSep's all-liquid, gel-free format, samples of intact proteins are available to customers for use with further investigative techniques.  A map displaying the global protein profile of a sample as well as comparative mapping analysis to study protein-protein and protein-drug interaction, are two of the protein expression profiles available.

--Abstract forthcoming--

"SPR/MS: An Approach to Protein Characterization and Identification"

Joanne Bruno, Biacore Incorporated jbruno@biacoreinc.com

In an era when Proteomics promises to yield a comprehensive understanding of proteins and their roles, Biacore’s surface plasmon resonance (SPR) based instruments are poised to play a central role in addressing all aspects of proteome analysis.  Biacore instruments facilitate protein separation and identification and provide real-time, quantitative, functional information in a reproducible manner. Biacore provides a versatile and powerful approach to Proteomics particularly because of the ability to complement current techniques, such as 2-D gels and mass spectrometry. The combination of SPR and mass spectrometry has increased the opportunity for identification and secondary characterization of binding partners through the use of a single analytical tool. The present study demonstrates the latest techniques for integration of Biacore with Mass Spectrometry.