ORAL PRESENTATIONS

Phil Andrews

Jens Coorssen  
University of Calgary         

Although 2D-PAGE continues to offer the highest resolution separation of proteins, it is somewhat dogmatically considered to be of more limited use in the analysis of hydrophobic membrane proteins.  Through systematic testing and optimization of associated protocols, from sample extraction through to staining and image analysis, we have found marked overall improvements in the resulting protein maps.  Importantly, this highly reproducible, routine protocol yields very high resolution separations of membrane proteins, enabling a new level of rigour in proteomic analyses.  In parallel, we have refined semi-dry transfer and high sensitivity immunodetection protocols to more fully enable simple, quantitative analyses of specific (low abundance) proteins.   We have also addressed the issue of proteins that remain poorly resolved in the standard 2D-PAGE separation format, including proteins stacked at pH extremes, unresolved peptides migrating at the separation front, and areas of the gel obscured by high abundance proteins.  We have used highly refined third electrophoretic separations to significantly improve the resolution in these traditional problem areas.  Importantly, these gains were achieved without detriment to the quality or resolution of the 2D gel itself.  Overall, this combination of methods represents a range of simple, reliable and cost-effective techniques for rescuing data (e.g. proteins) frequently lost in more traditional 2D-PAGE approaches. Our approach requires neither ‘retooling’ nor more specialized consumables than required for 2D-PAGE.  We are currently applying this optimized 2D-PAGE approach to the dissection of molecular mechanisms, including a translational research project that demands highly reproducible analyses of limited clinical samples. 

Jeff Smith         
York University 

The ciliated protozoan Tetrahymena thermophila is a unicellular eukaryote characterized by nuclear dimorphism, with a degree of cellular structural and functional complexity comparable to that of human and other metazoan cells.  It has proven extremely valuable as a model organism for many genetic and molecular biological studies, and has recently had its genome sequenced (http://www.tigr.org/tdb/e2k1/ttg/).  Two dimensional LC separations of a tryptic digest of the ciliary proteins of T. thermophila have been conducted using SCX followed by RP LC using a LC Packings Nanobore LC system; the RP LC eluent was directly infused into a QSTAR Pulsar instrument for ESI MS/MS analysis.  MS/MS data were searched in-house using Mascot software equipped with a complete proteome database made by translating a series of combined contiguous sequences, named scaffolds, in six reading frames.  Scaffold translations were treated as large proteins (2 – 75 MDa), and matching peptides localized in close proximity to each other indicated a coded region of the translated genome; these regions were BLASTed to find homologous proteins, and thus annotate the hit.  Replicate analyses have confirmed the identification of 234 proteins arising from distinct locations of the genome.  Many of these proteins are identified by strong mass spectral evidence, yet show little to no similarity to any known proteins.  Significantly, the genomic locations encoding this latter class of proteins are identified in this approach; this provides the necessary information for implementing downstream experimentation, such as knockouts and other biological strategies available for Tetrahymena, to discover protein functions.  Additionally, this class of proteins has been searched against the NCBI nt database to discover previously unidentified ORFs.  We have also used EST data available to annotate intron locations in genomic sequence of axonemal proteins with matches to Tetrahymena ESTs.  Finally all sequences have been compared to the proteome of the non-ciliate yeast.

The presentation will focus on recent work from our group, describing how the combination of on-line rapid mixing methods with electrospray mass spectrometry (ESI-MS) can be applied to monitor the dynamics of biological processes in solution. This approach allows kinetic studies to be carried out in the time range of milliseconds to minutes, and it provides direct information on protein conformation, ligand binding, and protein-protein interactions. Additional insights into protein structural dynamics can be obtained by applying on-line isotope exchange methods. The principles underlying these methods will be highlighted in studies on the subunit disassembly and unfolding of inducible nitric oxide synthase (iNOS). Kinetic measurements by ESI-MS provide detailed insights into the mechanism of this process, which involves a number of previously undetectable short-lived intermediates. In addition, the presentation will discuss recent advances in the use of hydrogen/deuterium exchange methods for exploring structural changes of enzymes during catalysis.

Arsalan Haqqani
IBS-NRC, Ottawa          

Ischemic stroke is caused by a localized deprivation of blood supply to brain tissue. Brain microvessels are lined by endothelial cells that display a tightly-sealed blood-brain barrier (BBB) phenotype. Ischemic stroke rapidly initiates a series of biochemial and molecular processes that disrupt BBB leading to brain edema and inflammation. The exact molecular mechanisms that lead to BBB disruption, including changes in gene and protein profiles occurring in the microvessels are not known. Laser-capture microdissection (LCM) is a powerful technology for dissecting a selective population of cells and multicellular structures from heterogeneous tissue specimens. We demonstrate the use of isotope-coded affinity tags (ICAT), an emerging proteomic technique, in combination with LCM to analyze a small number of multicellular microvessels dissected from rat brain tissues from an in vivo stroke model.  An in vitro model of ischemic stroke was also examined using ICAT.  Proteins extracted from the two models were ICAT-labeled and analyzed by mass spectrometry. We have developed in-house softwares with built-in normalization methods to analyze a large quantity of data in a more efficient manner than commercial softwares.  Results using this method have identified changes in several proteins in both models that are consistent with the physiological and pathological changes observed after stroke, e.g., changes in inflammation-associated and stress-induced proteins, signal transduction molecules, transporters and extracellular matrix proteins. Induction of several proteins has been validated by various biochemical methods.

Jack Greenblatt
University of Toronto         

We are systematically carrying out tandem affinity purifications from yeast strains with C-terminal, intrachromosomal TAP tags and identifying both the subunits of the purified protein complexes and weak interactions between protein complexes by SDS-PAGE/MALDI-TOF mass spectrometry and gel-free tandem mass spectrometry.  Purifications have been carried out thus far from approximately 4500 different tagged strains encompassing nearly all the soluble proteins of S. cerevisiae.  Our major current objective is to systematically organize these protein complexes into functional pathways for many of the large and important areas of yeast biology, including transcription by RNA polymerase II, protein trafficking in the ER and Golgi, DNA metabolism, RNA metabolism, cell cycle, and mitochondrial proteins. Genes are assembled for any given category on the basis of annotations and the literature, localization, synthetic genetic interactions between deletion mutants, gene co-regulation data, and protein co-purification. For all the 200-500 non-essential genes in any given category, synthetic genetic array (SGA) technology is used to create all possible double deletion mutants and growth rates are assessed in an automated fashion in order to identify synthetic growth defects or synthetic lethality.  Hierarchical clustering is then used to group proteins into functional pathways based on the principle that genes in the same pathway should have similar sets of synthetic genetic interactions. Validated examples of the application of these technologies to transcription and DNA repair will be presented.        

Bin Ma 
University of Western Ontario   

Tandem mass spectrometry (MS/MS) is the most reliable way to identify proteins and peptides.  Two computational approaches, database search and de novo sequencing, exist for interpreting the MS/MS data.  The former matches uninterpreted MS/MS spectra with peptides/proteins in a protein database and reports the best matching peptide/protein.  The latter computes the peptide sequences directly from the MS/MS data, without the help of a protein database.  Because scientists often work on novel proteins or species whose genomes are not known, the de novo sequencing approach is the only alternative   in these circumstances.

Because of the importance of the de novo sequencing problem, many de novo sequencing software packages have been developped.  These are represented by Lutefisk 1,2 and PEAKS 3,4.   In addition, mass spectrometer manufacturers have also developed their own de novo sequencing software, such as PLGS from Micromass and BioAnalyst from MDS Sciex/ABI.   Previous comparisons made by several different groups suggested that PEAKS (version 2.3 and earlier) performed the best in many test cases 3,5,6.  This presentation describes a new technique used in PEAKS version 2.4 that significantly improves PEAKS’ de novo sequencing.

"The Complimentary Role of High Density Tissue MicroArrays (TMA’s) In Protein Expression Profiling Of Human Endometrial Cancer"

Terence Colgan
Mt Sinai - Toronto         

Protein expression profiling and identification using both whole tissue homogeneates and microdissected tissue of endometrial cancer (EmCa) have revealed a number of potential cancer markers (PCM’s). One PCM, calgranulin, A had been detected in whole tissue lysates using SELDI-MS and identified using shotgun nanoLC/MS/MS, but its precise origin remained unclear. This question provided a good opportunity to assess the value of high-density tissue microarray (TMA) in protein expression profiling of disease. The construction of a TMA permits immunohistochemical (IHC) staining of multiple benign and malignant tissues under identical technical conditions, and efficient microscopic examination and scoring. A pilot TMA was built for immunohistochemical (IHC) studies to assess whether this technique could facilitate the localization and site of origin(s) of PCM’s.

Methods: A single 32 core TMA consisting of physiologic and malignant human endometrial tissue from formalin fixed paraffin embedded tissue blocks was constructed, and IHC staining for calgranulin A performed.  Immunostaining in three tissue compartments (macrophage/granulocyte, epithelium/carcinoma, and stroma/myometrium) was assessed, and the intensity of any staining graded semi-quantitatively (0 to 3+).

Results: Calgranulin A was detected in malignant epithelium (1 to 3+ intensity) in one half of malignant cases. In contrast, calgranulin A was detected in benign epithelium in only 1 of 10 cases. Macrophages and granulocytes of both malignant and benign endometria also demonstrated strong calgranulin A positivity; stroma and myometrium were uniformly negative.

Conclusions: This TMA-IHC study of calgranulin A demonstrates the capabilities of TMA’s in protein expression profiling of human cancers. The TMA-IHC study for calgranulin A permitted: 1. confirmation of the identity of the PCM derived by prior MS investigations, 2. localization and semi-quantitation of the PCM in malignant tissues, and 3. detection of the PCM within other non-malignant tissues. It is concluded that TMA’s have a complimentary role to other proteomic techniques in the discovery and study of novel PCM’s in human cancers.        

John Brennan    McMaster University     

An emerging method for screening of compound mixtures is frontal affinity chromatography interfaced with mass spectrometric detection.  This method relies on interactions of ligands with an immobilized protein, with stronger binders being retained to a greater degree on the column.  We describe a newly developed capillary-scale, monolithic silica bioaffinity column format that is amenable to immobilization of a wide range of target proteins, including soluble and membrane-bound enzymes, regulatory proteins and membrane-bound receptors.  The columns contain a combination of small pores, which are able to entrap proteins, and larger pores that provide flow channels that allow column operation with low backpressure.  The columns are shown to be suitable for high-throughput screening of compound mixtures, in-line LC/MS analysis of enzymatic reactions, including inhibition of such reactions, and other studies involving small molecule:protein interactions.  The use of the bioaffinity columns for screening compounds using different LC/MS formats is also described. 

Andrew Emili
University of Toronto         

Gel-free shotgun peptide sequencing using high-throughput tandem mass-spectrometry has become a powerful method for analyzing changes in global protein expression patterns in cells and tissues as a function of developmental, physiological and disease processes. However, biologists are keen to gain additional insight into perturbations affecting protein function and/or the regulation of biochemical pathways. Current methods for investigating protein-protein interactions (functional proteomics) on a genome-scale are tedious, and are not generally applicable to dynamic biological settings. Hence, there is a great need for rapid and efficient functional proteomic methods that are broadly applicable to diverse biological settings. In this presentation, I will discuss the practical utility of well-established procedures of biochemical fractionation, combined with comprehensive shotgun sequencing, as a suitable platform for systematic functional proteomic profiling of complex biological samples.

Ken Yeung       
University of Western Ontario   

Ultra small-volume sample preparation techniques were developed using capillary electrophoresis to address the following two mostly encountered challenges in mass spectral analysis of trace level proteins.

1. Sensitivity.  It has been nearly five years since Richard Smith reported the zepto mole protein sensitivity of FTICR MS (Anal. Chem. 2000, 72, 2271-2279).  Yet, such ultra trace analysis remains practically challenging because it currently lacks the technique to effectively preconcentrate sample to nano- or subnano-liter volume, which is necessary to meet the concentration detection limit of zepto mole sensitivity.  The development of ultra low-volume preconcentration of proteins, from microliters to nanoliters, will be presented.  The technique is based on trapping of proteins at a junction of two buffers with different pH values.  Extreme preconcentration factors up to 1700 were obtained for 1 µg/mL myoglobin.

2. Ionization Suppression. Even the most advanced (expensive) mass spectrometry suffers the problems arise from ionization suppression.  To tackle this problem, selective isolation of specific peptides from a mixture based on the isoelectric point has been developed.  The procedure can be used to remove abundant components in a sample of microliter volumes, and thus allowing the detection of trace components without interferences.  We were able to detect low atto moles peptides (in nanoliter-volumes) from a sample background of other peptides at 100,000 times higher concentration.

Michael Siu
York University 

John Marshall   
Ryerson           

We prepared phagosomes from polystyrene beads engulfed by RAW macrophages and human neutrophils with or without IgG opsonization.   Over the time course of particle engulfment, neutrophils or macrophages were disrupted with a French press and phagosomes purified over sucrose gradients.  As a negative control, crude cellular lysates and culture medium were incubated with polystyrene beads.  The resulting proteins were prepared by micro chromatography followed by trypsin digestion and LC-MS/MS.  We found that proteins of the endoplasmic reticulum, histones and cytoskeletal proteins were associated with the negative control and were apparently cellular contamination of the phagosomal preparations.   No accumulation of endoplasmic reticulum markers was observed at the phagosome using transfection of GFP fusion proteins and microscopic measurements of RAW cells.  In contrast, many receptor associated signaling proteins, including the Fc receptor, and many of the class of signal molecules associated with the vesicular model of phagocytosis were observed by MS/MS and subsequent microscopy including the src, syk, p110, p85, ship-1 PLC, PLD, and the ras superfamily.  We collected MS/MS data mapping to specific ras, rac and rho proteins  and their respective GTPase activating and exchange factors such as ELMO, dock 180, and crk homologs.  Thus we found agreement between MS/MS and live cell confocal microscopic evidence in support of the vesicular model of phagocytosis.