Events

Title	Poster Abstract
“Haptoglobin ao1, New Ovarian Cancer Biomarker Identified by Mass Proteomic Chip Technology” by Bin Ye, Harvard Medical School, Bin_ye@hms.harvard.edu	The objective of this study is to identify potential biomarkers for the early detection of ovarian cancer in serum and to establish the technology for high throughput screening with combination of Surface Enhanced Laser Desorption/Ionization (SELDI)-Mass Spectroscopy (MS) and Liquid Chromatography (LC). With the SELDI ProteinChip arrays, we screened for protein markers of molecular weight less than 50 kDa in a total of 108 age-matched serum samples (58 cases and 50 normal controls). Several protein peaks were identified as potential markers for different histologic subtypes of ovarian cancer. One protein peak at about 11700.00 Da was identified from the profiles using the IMAC3 chip. This protein appeared in all histologic types of ovarian cancer cases, but not in controls. At a concentration intensity of greater than 0.2 from the SELDI output, the sensitivity and specificity of this biomarker for ovarian cancer was 83% and 70%, respectively. This protein was further purified by affinity chromatography and sequenced by Ion Trap Tandem Mass Spectrometry. It was identified as the one form of Haptoglobin að1ð chain. The elevated level of the Haptoglobin að in sera in ovarian cancer patients, suggested by SELDI, was confirmed by western blotting with the purified polyclonal antibody. The developed ELISA screening test also showed 82% sensitivity and 83% specificity in 94 cases and 99 normal controls. The elevation of Hp-að chain level in cancer patients is significant (P<0.0001) compared to those in benign cancer and other gynecologic cancer patients. Of interest, complete Haptoglobin has been previously been found to be elevated in ovarian cancer patients. Ours is the first report to suggest that use of the Hp-að1ð chain is a more sensitive marker. More detailed studies are necessary for practical application of Haptoglobin að chain as a marker in ovarian cancer early detection.
“LC MALDI-Tof MS of Peptide Mixtures using Continuous Eluent Deposition onto Pre-Coated MALDI Targets” by Steven Cohen, Waters Corporation steven_cohen@waters.com	We have investigated the potential advantages for MALDI-TOF MS provided by the continuous deposition of HPLC eluent onto the MALDI target. These advantages include increased sensitivity by pre-concentration of the analyte, separation of interfering contaminants from the analyte and increased dynamic range of peptide abundance that can be analyzed. A further advantage is that the chromatographic eluent is frozen in time on the sample plate and may be stored for analysis at a later time by other techniques. In these studies we have analyzed peptide mixtures separated by reversed phase LC and deposited the eluent continuously onto a MALDI sample plate pre-coated with -CHCA matrix. Both standard peptides and mixtures obtained from tryptic digestions of intact proteins have been analyzed. The initial protein mixtures vary in complexity from 4 standard proteins to ca. 75 ribosomal proteins. Standard proteins were mixed in different amounts to determine if low abundance proteins can be detected in the presence of high abundance proteins. Detection sensitivity is in the low femtomole range, and the dynamic range is at least two orders of magnitude. The ribosomal protein mixtures are separated using 1-D SDS PAGE and the gel bands are excised and digested with trypsin. All digests are then separated by reversed phase HPLC and collected onto MALDI targets. These targets are then analyzed using MALDI-TOF MS and MALDI-QTOF MS/MS. The peptide mass maps and peptide sequencing information that is generated by this method is used to determine the identity of the proteins therein. The peptide mass mapping results from the LC-MALDI work is compared to that obtained by the traditional sample loading methods. This work demonstrates that using the LC-MALDI to continuously sample a RP HPLC peptide separation onto a MALDI target plate provides for improved peptide mass mapping coverage over traditional spotting methods in terms of the sequence coverage and the dynamic range of proteins that can be identified from one mixture.
"SPR/MS an Approach to Protein Identification and Characterization" by Kara Herlihy, Ph.D., Biacore, Inc. Contributing Authors: Osten Jansson¶, Jos Buis¶, Andrei Zhukov¶, Kara Herlihy§ and JoAnne Bruno§ ¶Biacore AB, Uppsala, Sweden §Biacore, Inc., Piscataway, NJ USA kherlihy@biacoreinc.com	In an era when Proteomics promises to yield a comprehensive understanding of proteins and their roles, Biacores Surface Plasmon Resonance (SPR) based instruments are poised to play a central role in addressing many aspects of proteome analysis. Biacore instruments facilitate protein separation and characterization through provision of real-time, quantitative and functional information. Biacore provides a powerful and versatile approach to Proteomics particularly because of its 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. Surface plasmon resonance in combination with mass spectrometry (SPR-MS) can provide a means of selective binding, recovery and identification for specific proteins from complex biological mixtures. The latest techniques for integrating Biacore systems with mass spectrometry will be discussed. Additionally, multiple examples will be presented to demonstrate how these two technologies are combined in real life situations to yield maximum information in a minimum amount of time.
“Mass Spectrometric Identification of DNA-Binding Proteins and Computational Identification of Signal Transduction Networks” by Martin Steffen, Harvard Medical School steffen@rascal.med.harvard.edu	We have developed a method for isolating and identifying DNA-binding proteins from total cell lysates. Our method is based on in vivo cross-linking of DNA-binding proteins to DNA with formaldehyde, enrichment by equilibrium density centrifugation in cesium chloride, separation by 1D LC/MS/MS. Our studies in E. coli and cultured human cells reveal significant enrichment for abundant and and low-copy DNA-binding proteins. We have detected 25 DNA-binding proteins in E. coli, and 64 confirmed and 31 potential DNA-binding proteins in human 293 cells. We have developed a computational approach for identifying signal transduction networks in yeast. Our program, STNSearch computes paths based on published protein-protein interaction data derived primarily from large-scale two-hybrid studies, and gene expression data obtained from microarrays. In this approach, our algorithm draws all possible pathways through the yeast protein-protein interaction map, starting at membrane proteins and terminating at DNA-binding proteins. These potential pathways are then ranked based upon the similarity of the expression profiles of pathway constituents. Networks are determined entirely by the integration of the protein-protein interaction data with the microarray expression data, without prior knowledge of any pathway intermediates. We show that our technique accurately constructs three MAP Kinase signaling networks in yeast. This approach should enhance our ability to discover new signaling components and, more generally, allows one to browse two large whole-genome datasets and extract sub-networks comprised of proteins that are both highly interconnected and highly-correlated.
"Differential In-Gel Electrophoresis (DIGE), A Novel Way to Detect Changes in Protein Expression" by Christine Rozanas, Ph.D, Amersham Biosciences, Piscataway NJ chris.rozanas@am.amershambiosciences.com	The rapidly expanding field of proteomics relies heavily upon the use of 2-dimensional gel electrophoresis of protein samples followed by mass spectrometric spot identification. Amersham Biosciences has recently introduced a technology that allows one to run multiple samples on the same gel thereby eliminating many of the analytical errors associated with matching samples run on different gels. Up to three samples can be run on the same gel, or an internal standard can be used to simplify the statistical analysis of a multiple gel experiment using a student's T-test to determine statistically relevant biological changes. This new technology (DIGE) and its interface into the proteomics approach will be discussed. In addition preliminary data from a study comparing tomato fruit ripening between normal and mutant plants will be presented.
"Nano-scale Variable Flow Chromatography for High Sensitivity Proteome Studies" by James Langridge¹, Alan Millar¹, Chris Hughes¹, Hans Vissers¹, Tad Dourdeville², Bonnie Marmor³ and Philip Young¹ 1 Micromass UK Ltd, Manchester UK 2 Waters Corporation, Milford, MA, USA 3 Micromass Inc, 100 Cummings Center, Beverly, MA USA james.langridge@micromass.co.uk	Mass spectrometry has firmly established itself as the primary technique for identifying proteins due to its unparalleled speed, sensitivity and specificity. The most common strategy involves digestion of the protein to yield smaller stretches of peptide sequence. Complex peptide mixtures are typically separated by micro-capillary liquid chromatography followed by on-line mass spectral detection via an electrospray ionisation (ESI) source. The peptides are analysed using automated acquisition modes whereby conventional MS and (low energy) MS/MS spectra are collected in a data dependant manner. This information can be used directly to search databases for matching sequences leading to identification of the parent protein. However often the limiting factor for identification of the protein is the quality of the MS/MS spectrum produced. One very elegant solution to this problem is to use a chromatographic technique known as variable flow chromatography or "peak parking". In this experiment we can focus on a particular mass spectral peak of interest by reducing the flow rate from the usual 200nL/min to approximately 20-30nL/min. The precursor ion is held in the MS/MS mode for an increased length of time, such that a large increase in sensitivity can be obtained. Multiple experiments can be performed on several co-eluting species in an automated way by the Q-Tof mass sp ectrometer and the Micromass CapLC pump working in a synchronized manner. The combination of intelligent HPLC flow rate control and on-the-fly data directed analysis has been applied to the analysis of proteolytic tryptic digests enable the maximum amount of information to be extracted from a single acquisition. This approach will be discussed with examples of where this approach has been used for targeted proteomic experiments looking at post translationally modified peptides, in this case phosphorylation.