Poster

"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.
 

"The Use of LC-MALDI-MS and LC-MALDI-MS/MS as Methods Enhance the Investigation of Peptide Mass Fingerprint Spectra"

Richard Tyldesley, Micromass UK,  Richard.tyldesley@micromass.co.uk

Peptide mass fingerprinting (PMF) is a well-established method used to identify proteins extracted from two-dimensional electrophoresis gels. Identification is achieved by matching the masses of observed tryptic peptides against those theoretically derived from protein data banks. Improved sample separation techniques such as the deposition of the HPLC eluent directly onto the MALDI plate increases sensitivity, dynamic range and confidence of protein identifications. PMF spectra often include many other peaks originating from natural post-translational modifications (PTM), modifications introduced during sample processing, sample contamination or less specific proteolytic digestion products.

A variety of protein standards were digested by trypsin and continuously eluted via a reverse phase HPLC column on to a MALDI sample plate (pre-coated with CHCA matrix). The sample plates were initially analyzed using axial MALDI-TOF-MS and the peptide spectra generated were used to identify the protein standard by peptide mass fingerprinting. Spectral peaks observed in the MS spectrum, which could not be attributed to classical tryptic peptides or typical modifications associated with the protein analyzed, were subsequently sequenced by CID MS/MS experiments using an orthogonal MALDI-QTOF instrument.

"Novel, High-speed, Multicolumn Processes at 
the Nanoliter Scale"

Doug Boyd, Gyros Corporation 
doug.boyd@gyros.com

Peptide mapping by MALDI mass spectrometry is frequently used for protein 

identification in proteomic studies and effective sample preparation is 

critical in achieving high sensitivity mass spectra and successful protein 

identification. However, sample preparation for MALDI MS analysis often 

becomes a rate-limiting step in the identification process. Conventional 

preparation techniques with multi-step processes increase the risk of 

sample loss so reducing the sensitivity of the final analysis and care 

must be taken to ensure reproducibility and reliability of the results.

Gyrolab MALDI SP1 is an application-specific CD microlaboratory in which 

protein digests are concentrated, desalted, eluted with matrix and crystallized 

onto MALDI target areas within the CD. Each step in this integrated process 

has been optimized to enhance sample recovery and produce homogenous crystals 

in a reliable manner. MS analyses from samples prepared on Gyrolab MALDI 

SP1 demonstrate sensitivity levels in the attomole-femtomole range with 

a high level of reproducibility.

To further enhance reproducibility, 96 samples can be processed simultaneously 

under uniform conditions in each CD. CDs are run in Gyrolab Workstation 

under the control of software that includes a preprogrammed method for 

Gyrolab MALDI SP1. Each CD is then transferred to a MALDI mass spectrometer 

for analysis. Sample preparation on Gyrolab MALDI SP1 has yielded higher 

sensitivity results in comparison with standard pipette tip purification 

technology.

"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.
 

"Comparing the Proteome in the Cytosol Fraction of Drug-resistant and Non-resistant Breast Cancer Cells"

Yetrib Hathout, University of Maryland,     hathout@wam.umd.edu       

In the last few years proteome analysis has been widely used to study differential protein expression between normal and cancer cells. However, very few studies have compared the proteome of drug resistant and drug susceptible cancer cells.  In all published studies attention has been focused on proteins that are up-regulated in acquired drug resistance. In the present work differential protein expression is examined with special attention to down regulation in the cytosol fraction of a breast cancer MCF7 cell line selected for resistance toward the alkylating agent melphalan. The strategy used is a combination of cell fractionation, two dimensional gel electrophoresis (2-DE), mass spectrometry and bioinformatics.

Wild type MCF7 and melphalan resistant MCF7 cells were grown to confluence in serum supplemented media. The cells were harvested by trypsin and the cytosolic fraction was obtained by differential detergent fractionation using a digitionin/EDTA solution containing protease inhibitors. The cytosolic proteins from the two cell lines were separated by 2-DE. The 2-DE images were compared and the spots of interest were excised and digested with trypsin. The resulting peptides were analyzed by MALDI-TOF-MS and/or nano-spray Qq-TOF-MS.  Database search was performed by entering the MALDI-mass list in the Mascot search engine or by sequence tags obtained on the peptides following nano-spray MS/MS analysis.

From the excised spots 100 proteins were successfully identified and annotated in the 2-DG map of the cytosolic fraction of MCF-7 cells. For the first time cyclophilin A is reported to coexist as an N-terminally acetylated and non-actylated form in mammalian cells. Comparison of multiple digitized gel arrays detected several spots as candidates for differentially expressed proteins in melphalan resistant MCF-7 cells. The up-regulated proteins included retinoic acid binding protein II, an isoform of the macrophage migration inhibition factor and other unidentified proteins. The down-regulated proteins included calreticulin, cyclophin A, and an isoform of the 27 kD heat shock protein.  Correlation of the differential expression of some of the proteins with acquired resistance of MCF7 cells to melphalan is discussed.
 

Marion Gehrmann, University of Maryland,
gehrmann@wam.umd.edu

Development of multi-drug resistance is the main reason for the failure in breast cancer chemotherapy. Differentially expressed proteins are directly or indirectly correlated to the drug resistance of the cancer cell. Thus, comparative studies of protein levels in drug-susceptible and -resistant human breast cancer cells can give an insight into the mechanisms of resistance as well as into the understanding cross-resistance. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) provides an easy tool to study differential protein expression in drug-resistant and drug-susceptible breast cancer cells. However proteins have to be mapped prior to any comparative studies using 2-DE. Even though proteomics enables to study all cellular proteins simultaneously, the analysis of whole cell extracts is difficult, since high abundant proteins can mask low abundant ones making their detection difficult if not impossible. Pre-fractionation reduces the complexity of the samples. In the present work the cytosolic fraction of MCF-7 breast cancer cells was examined, using a combination of 2-DE, mass spectrometry and bioinformatics.

The cytosolic fraction of MCF-7 breast cancer cells was obtained by differential detergent fractionation using digitonin. Cytosolic proteins were then separated by 2-DE, excised and digested with trypsin. The resulting peptides were analyzed by MALDI-TOF-MS and/or nanospray Qq-TOF-MS. Database search tools like TagIdent and Mascot were then used to identify the protein precursors of these peptides, based either on the peptide mass lists obtained from MALDI or on sequence ions in the tandem mass spectra.

To date 110 proteins have been identified and mapped on the 2-DE gel of the cytosolic fraction of MCF-7 cells. Several  post-translational modifications, such as phosphorylation and acetylation, have been determined by peptide sequencing using nanospray-MS/MS analysis. Adenine phosphoribosyltransferase and cofilin-1 were both found to be N-terminal acetylated, while the database reports both proteins without possible acetylation.  Heat shock protein 27kDa, elongation factor 2 and cofilin-1 coexist in two isoforms, a phosphorylated and a non-phosphorylated form.

"Characterizing, Interpreting and Understanding the Results of 2D Gel Experiments"

Robert Dunkle, Scimagix,    rdunkle@scimagix.com

An automated registration, analysis and pattern mining system to store, analyze and search the visual content of 2D gels was developed by Scimagix in a partnership with Pfizer Global R & D. The system alleviates the problems in spot detection and alignment to enhance 2D gel analysis and by enabling the creation of a 2D gel knowledge base that can be mined. The system is in use at Pfizer and other companies; Pfizer’s outcomes using this system for rapid assessment of mechanism of action (MOA) is described.

Refinements in 2D electrophoresis gel (2D gel) technology and mass spectrometry (MS) have made proteomics investigations an increasingly common component of progressive discovery programs. To capitalize on the images and data generated organizations must alleviate problems with spot detection, alignment and image/data storage. The opportunity is to make this data accessible and searchable, with the goal to enable the retrieval of gels with similar protein expression patterns using systems that can store and search the visual content of 2D gels. Pfizer Global Research & Development (Ann Arbor, MI) partnered with Scimagix, Inc. (San Mateo, CA) to develop such a system – this system is now in use by Pfizer and other of the top pharmaceutical companies.

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.