POSTER PRESENTATIONS

Crystallization Data Mining: Knowledge-Based Protein Crystallization Screen Design

Chunmin Li, University of British Columbia

chunminli@shaw.ca

Crystallization of a biological macromolecule is the first and most difficult step towards its structural determination by X-ray diffraction.  Although close to 24,199 biological macromolecular crystal structures have been deposited in the Protein Data Bank, very little has been published in the literature demonstrating that crystallization information has been used to develop predictive models.  Most of the initial screens are still empirical and random.  To gain insight into how to overcome the problems associated with protein crystallization, we have initiated a project to study correlations between the properties of proteins targeted for structural studies and successful crystallization conditions. The results revealed that scientists can design a more successful crystallization screen by using protein information than by simply following the global crystallization success distributions derived for all proteins.  In this way, we can significantly reduce the cost and material requirements in structural genomics.

Comparison of Plasma Proteomes of Different Species

David D. Y. Chen, University of British Columbia

chen@chem..ubc.ca

Choosing appropriate animal models for the development of pharmaceutical drugs for human illness is often a challenge in research labs and pharmaceutical industry.  This work demonstrate that 2-dimensional protein fractionation systems can be used to quickly compare the proteomes of the tissues or blood of the potential animal models with that of human to aid the animal model selection process.  With a Beckman Coulter ProteomeLab� PF-2D system, human blood proteome is compared with that of canine. Some significant differences between the two proteomes can be observed.

Ultrasensitive Proteome Analysis Of Deinococcus Radiodurans

Emily H. Turner, University of Washington

emilyt@u.washington.edu

Deinococcus radiodurans are highly radio-resistant bacteria with unique DNA protection and repair mechanisms. Upon exposure to high levels of ionizing radiation, D. radiodurans appears to sustain little permanent DNA damage, apparently due to rapid recovery responses. Using capillary electrophoresis, our group is performing proteome analysis of D. radiodurans to track changes in protein expression following exposure to ionizing radiation.

Capillary electrophoresis is especially suited to the analysis of biomolecules. Our group has developed two-dimensional capillary electrophoresis (2D-CE) to perform proteomic analysis. Two modes of capillary electrophoresis are combined in this technique: capillary sieving electrophoresis (CSE), and micellar electrokinetic chromatography (MEKC). In the first dimension CSE separates proteins based on their molecular weight; in the second dimension MEKC separates the proteins by hydrophobicity. Unlike traditional two dimensional SDS-PAGE gels, 2D-CE is rapid and fully automated; separations are complete in less than two hours. Laser-induced fluorescence detection provides low zeptomole (10^-19) limits of detection, permitting single-cell analysis.

Cell extracts are run on 2D-SDS-PAGE gels, and spots identified with mass spectrometry. Identified proteins are spiked into 2D-CE runs for peak identification. Single-cell 2D-CE will be used to track stochastic gene expression and changes in protein profiles after exposure to ionizing radiation.

Active-site Peptide ‘Fingerprinting’ of Glycosidases in Complex Mixtures by Mass Spectrometry: Discovery of a Novel Retaining beta-1,4-Glycanase in Cellulomonas fimi

Omid Hekmat, University of British Columbia

ohekmat@chem.ubc.ca

New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion.  Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of beta–retaining endo-glycosidases in complex proteomes.  Using a biotinylated, cleavable, 2-deoxy-2-fluoro-xylobioside inactivator, we have isolated and identified the active-site peptides of target retaining beta–1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes and the secreted proteome of the aerobic, mesophilic soil bacterium Cellulomonas fimi.  The active-site peptide of a novel C. fimi beta–1,4-glycanase was identified in this manner. The peptide sequence, which includes the catalytic nucleophile, is highly conserved among members of glycosidase family 10 and hence acts as a ‘fingerprint’ of the target enzyme in the proteome ‘population’.  PCR-amplification, using primers based upon the identified sequence, revealed a novel glycanase with about 80% sequence homology with xylanases from Streptomyces sp.

Development of a Sheath-Flow CE-MS System for Peptide Analysis

Regine M. Schoenherr, University of Washington

regimens@u.washington.edu

Capillary electrophoresis (CE) was first coupled with mass spectrometry (MS) in the late 1980s¹.  One of the interface designs for this coupling is a sheath-flow interface, which supplements the effluent from the capillary with sheath liquid to obtain a more stable electrospray than is sometimes achieved without a sheath liquid.  CE-MS is very suitable for protein and peptide analyses due to the speed and efficiency of CE separations and the inherent identification capability of MS. 

In the presented work, an in-house built CE instrument has been coupled via a sheath-flow interface to a triple quadrupole-linear ion trap hybrid mass spectrometer.  The capillaries have been modified with a poly(vinyl alcohol) (PVA) coating to avoid band broadening caused by interactions between the capillary wall and the peptides.  So far, standard peptides and digests of standard proteins have been analyzed with the CE-MS system in MS and MS/MS mode. A preliminary estimate of the detection limit of the system is in the mid-attomole range. 

In the future, a microreactor will be added in front of the separation capillary.  Mostly, proteins are digested in bulk solutions to yield peptides for CE-MS analysis.  In addition to long digestion times, autodigestion of the enzyme might complicate the sample.  Therefore, it would be desirable to digest proteins on-line in a microreactor before the peptides are separated with CE.  Autodigestion is almost eliminated in microreactors that have enzymes immobilized in them, which allows a high concentration of enzymes in microreactors, resulting in very short digestion times.

The Human Protease CLIP-CHIP: Genomic Analysis Of All 715 Human Protease And Inhibitor Gene Transcripts In Human Breast Carcinoma

Reinhild Kappelhoff

reinhild@interchange.ubc.ca

Matrix metalloproteinases (MMPs) are an important family of proteases that correlate with tumor grade. MMPs traditionally were thought to degrade the ECM in metastasis, but the large number of bioactive mediators that are precisely processed by MMPs reveals MMPs to be important signaling proteases. Although MMPs are believed to be pivotal in cancer progression and metastasis, they are expressed in combination with other proteases and inhibitors, in which their expression levels are partial determinants of their relative importance. The availability of the human genome sequences has enabled the identification the degradome—the complete repertoire of proteases produced in man. By using primary information retrieved from public and private sequencing projects, together with data from the MEROPS, InterPro and Ensembl databases we have annotated a total of 558 genes encoding proteases or protease-homologs in the human genome (21 aspartate, 148 cysteine, 184 metallo, 177 serine, 28 threonine) and 157 inhibitors (Puente et al Nature Rev Genetics 4, 544-558, 2003). To analyze the behavior of proteases and inhibitors the human protease CLIP-CHIP was designed and constructed. The CLIP-CHIP is a dedicated chip containing all 715 human protease and inhibitor sequences presented by unique 70-mer oligonucleotides.

Work is in progress to analyze the protease degradome in human breast carcinomas (invasive ductal carcinoma). Following RNA labeling using different dyes for tumor and normal tissue, they were pooled and hybridized with the CLIP-CHIP. By analyzing the fluorescence intensity of the two dyes the increase or decrease of the expression level of all proteases and inhibitors were determined on a system-wide basis. The design of the dedicated protease CLIP-CHIP for the system-wide analysis of patterns of expression and activity of human proteases is a potent new tool for the analysis the role of proteolytic activity in cancer initiation and progression.    

“Quantitative proteomic investigation of MMP inhibitor side-effects in human breast carcinoma using Isotope Coded Affinity Tag and Tandem Mass Spectrometry” 

Georgina S. Butler

gsbutler@interchange.ubc.ca

Matrix metalloproteinases (MMP) are a family of 23 zinc endoproteinases. Since MMPs are collectively able to degrade all components of extracellular matrix, and for tumours to metastasise, i.e. to escape their encapsulation, move through tissues and into and out of blood vessels, extracellular matrix degradation is required, MMPs were targeted for inhibition as therapy in cancer patients. The most potent MMP inhibitors, the hydroxamate-based inhibitors, were evaluated in clinical trials, but these were relatively unsuccessful due to side-effects such as joint pain and myalgia which limited the dosage, as well as low degree of efficacy. The cause of these troublesome side effects has not been determined. Recently it has become clear that in addition to extracellular matrix molecules, MMPs cleave an array of bioactive molecules, including growth factors, cytokines, chemokines, cell surface receptors, growth factor binding proteins, protease inhibitors and adhesion molecules. With such a diverse range of molecules modified by the MMPs, each with downstream effects, it is not surprising that the cause of the side effects was not determined using conventional techniques. To tackle this daunting task, we are using Isotope-Coded Affinity Tagging (ICAT), a quantitative proteomics approach, to identify soluble, membrane and matrix proteins which are affected by treatment of a human breast cancer cell line (MDA-MB 231) or myofibroblasts with a hydroxamate inhibitor. Many of these protein “hits” are known  substrates of MMPs, which validates the method, and many are likely to be novel substrates.  These proteins will be further characterised to determine whether they could be responsible for the side effects.

Identification of Novel MT1-MMP Substrates Using Quantitative Proteomics–Isotope Coded Affinity Tag and Tandem Mass Spectrometry

Eric M. Tam

Through the hydrolysis of peptide bonds, proteases control critical biological processes in almost all aspects of biology. Thus, protease substrate identification is crucial in understanding all the complex roles proteases play in vivo. The identification of all substrates for an individual protease or a protease family is a technical challenge.  We used Isotope Coded Affinity Tag and protein sequencing by tandem mass spectrometry (MS/MS)  to quantify the levels of secreted or shed extracellular proteins in the media of MDA-MB-231 breast carcinoma cells transfected with membrane type 1-matrix metalloproteinase (MT1-MMP), a membrane bound member of the matrix metalloproteinase (MMP) family, or vector alone. Extracellular proteins displaying differential levels in MT1-MMP overexpressing cells versus vector transfected cells were digested with recombinant MT1-MMP. Protein cleavage was analyzed by SDS-PAGE, western blotting, N-terminal sequencing and MALDI-TOF MS. We report that IL-8, the serine protease inhibitor SLPI, the TNF receptor death receptor-6, pro-TNF-, and CTGF are previously undescribed substrates of MT1-MMP.

Degradomic discovery of novel matrix metalloproteinase-2 substrates by isotope coded affinity tag labeling and tandem mass spectrometry

Richard A. Dean

rdean@interchange.ubc.ca

Matrix metalloproteinase (MMP) 2 is an important protease in the 23 member family of proteolytic enzymes that collectively are capable of degrading all components of the extracellular matrix (ECM). Recently we showed that MMPs also cleave a wide array of bioactive molecules, including cell surface receptors, adhesion molecules, chemokines, cytokines, growth factors and protease inhibitors (Tam et al 2004 PNAS 101, 6917-6922). The aim of this study was to discover novel MMP-2 substrates using quantitative proteomic technology in order to better understand the in vivo physiological roles of MMPs. Methods: Murine fibroblastic cells immortalized from MMP-2 knockout mice were stably transfected with the active human MMP-2 gene (MMP-2 minus its propeptide) or the catalytically inactive E375A variant.  After 72 hours, phenol red free serum free conditioned medium was collected from each cell type and concentrated to ~3 mg/ml. Isotope-coded affinity tag (ICAT) labeling and multi-dimensional liquid chromatography inline with tandem mass spectrometry was used to identify cell proteins shed from the cell surface or the pericellular matrix, and extracellular proteins that were degraded or processed after transfection with the active human MMP-2.  Biochemical cleavage assays utilizing recombinant proteins were used to confirm specific proteomic hits, with MALDI-TOF mass spectrometry and EDMAN sequencing being used to determine the MMP-2 cleavage sites. Results: A number of known MMP-2 substrates were detected by the ICAT analysis validating this novel approach including: Collagen alpha 1 (I), (IV), collagen alpha 3 (IV), laminin alpha 5, fibronectin and SPARC. However a larger number of previously unidentified substrates were discovered and confirmed by biochemical assays: Connective tissue growth factor, Thrombospondin 1, Follistatin-related protein 1, Pleiotrophin and the cysteine protease inhibitor Cystatin C. Cleavage of Cystatin C removed the first 9 N-terminal amino acids resulting in a decrease in its protease inhibitory function towards Cathepsin L. Conclusion: The novel use of ICAT with tandem MS to identify new protease substrates extends the use of this degradomic procedure to explore the role of secreted proteases in complex milieu. Our work reveals that MMPs are important signaling proteases involved in the precise processing of many bioactive molecules in addition to cleavage of ECM components in physiological and pathological processes.

Oded Kleifeld

oded@interchange.ubc.ca

Matrix metalloproteinases (MMPs) are a family of homologous enzymes that play a leading role in the catabolism of the macromolecular components of the extracellular matrix and processing of bioactive molecules in a variety of normal and pathological processes. Understanding of MMP protease and protease-substrate repertoires expressed at specific time by a cell, tissue or organism is crucial to determine which of these are most important for disease-related or normal biological processes.

To reach that goal we are developing new tools for protease activity profiling to distinguish active enzymes from their inactive-precursor or inhibitor-bound forms.

We have engineered a novel probe that can be used to specifically track activity of MMP-2. This probe is based on the MMP-2 inhibitory of region of ?-amyloid precursor protein (APP) (Higashi S. and Miazaky K. /JBC/ 14020-14028, *278* 2003) combined with an affinity-tag. By using this probe we were able to affinity purify and monitor changes in active MMP-2 levels in crude protein mixtures.