EGFR(Salipro)

The seventh in the series of ETP Symposia (see Rapid Communications in Mass Spectrometry 2012, 26, 1515–1518 for a description and report on the 6th Symposium) was held at the Metro Toronto Convention Centre, Toronto, Canada, on 30 April to 1 May, 2013, and was chaired by Professor Daniel Figeys, Ottawa Institute for Systems Biology, University of Ottawa. The purpose of these symposia is to convene meetings of scientists and engineers from universities, government laboratories, industry and manufacturers of scientific instrumentation, to discuss novel technologies and methodologies applicable to research in molecular biology. The speakers and the Abstracts of their presentations are listed alphabetically below. The program illustrates some significant changes from earlier versions (www.etpsymposium.org) that were devoted almost entirely to proteomics (the 'P' in 'ETP'). In addition to new techniques and applications related to that area, this symposium featured work related to nucleic acid characterization, biomaterials discovery, water disinfection byproducts, and metabolomics, as well as more fundamental developments in mass spectrometry (ionization techniques and quadrupole theory) and sample preparation. The symposium is now more truly international, with speakers from Canada, USA, Europe and China. It is hoped that the wide variety of topics and backgrounds of participants, together with the highly informal setting, will help promote cross‐fertilization of ideas and unforeseen collaborations.Perdita E. Barran, Department of Chemistry, The University of Edinburgh, Edinburgh, UKResolution of solution structures in the absence of solvent (Invited Talk)Gentle application of nano‐electrospray to proteins buffered in solution to an appropriate pH allows us to use mass spectrometry to interrogate their solution conformations. This can be achieved directly with ion mobility mass spectrometry where we measure the rotationally averaged temperature dependent collision cross section of mass separated ions, or more indirectly with the use of dissociation methods. Data obtained from both methods provides insight to the structure and stability of the protein, and also detail on its interactions, especially when combined with atomistically resolved data from crystallography and/or computational approaches. Data were presented from two systems, the first was a peptide level model of the interaction between the transcription factor c‐MYC and its partner MAX; the second was a detailed structural evaluation of the metamorphic protein Lymphotactin.Ronald Beavis, Beavis Informatics Ltd, Winnipeg, CanadaFalse negatives in proteomics (Invited Talk)Proteomics experiments using tandem mass spectrometry to identify peptides and proteins are prone to various types of false negative identifications that can affect the utility of the information generated from the experiments, as well as their reproducibility. This talk discussed the classification of the most common types of false negatives as well as how they can be detected, remedied or avoided.John D. Brennan, Blake J. Helka and Elna D. Luckham, Department of Chemistry, McMaster University, Hamilton, CanadaImaging MALDI MS/MS of microarrays as a platform for high throughput biomaterials discovery (Invited Talk)This presentation highlighted recent work within the Biointerfaces Institute at McMaster University in the area of high throughput screening of biomaterials, with particular emphasis on the use of imaging MALDI for characterization of biomaterial microarrays. Using robotic handling systems and a combination of contact and noncontact microarray printing, we are able to produce several thousand biomaterials per day with a wide range of chemical compositions. Using sol–gel based materials as an example, the presentation showed the workflow utilized to develop new bioactive sol–gel materials for biosensing and small molecule screening applications. This includes methods to produce several thousand materials very rapidly, tools for rapid screening to identify " hits" that show a desired property (high biological activity, low non‐specific binding, etc.), and further detailed material analysis using a range of imaging methods based on fluorescence, XPS, MALDI‐MS/MS and SPR to fully characterize the properties of biologically active materials. In particular, this presentation emphasized the unique capabilities of MALDI‐MS/MS for evaluating biomaterial properties and the interaction of materials with biological samples. Methods for cataloging, mining and analyzing large datasets within the Database of Biointerface Interactions were also discussed.Rui Chen, H. Zou and D. Figeys, Ontario Institute for Systems Biology, University of Ottawa, Ottawa, CanadaCharacterization of cell membrane N‐glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC/MS/MSGlycosylation of membrane proteins plays important roles in cellular behaviours such as cell‐cell interaction, immunology recognition and cell signalling. Despite their importance the effective extraction of membrane protein, selective isolation of glycopeptides and mass spectrometric characterization of glycosylation are challenging current analytical techniques. In this study, a systematic approach was developed which combined an integrated hydrophilic interaction chromatography solid phase extraction (HILIC SPE) for simultaneous detergent removal and glycopeptides enrichment, and mass spectrometric identification of both protein N‐glycosylation sites and site‐specific glycan structure. The HILIC SPE condition was optimized to enable the use of high concentration of strong detergents, such as SDS and Triton X‐100, to dissolve highly hydrophobic membrane proteins, thus increasing the yield of membrane protein extraction. We illustrated the performance of this approach for the study of membrane protein glycosylation from human embryonic kidney cell lines (HEK 293T). 200 µg total protein digest was processed using this approach, leading to the identification of 813 N‐glycosylation sites from 568 proteins within two experimental replicates. Furthermore, 177 glycopeptides representing 82 N‐glycosides with both glycan composition and peptide sequence were identified by high energy collision dissociation.Andrew Crowell, A. Doucette and S. Rudolph, Department of Chemistry, Dalhousie University, Halifax, NS, Canada.Disposable two‐stage spin cartridges for protein purification in a top‐down proteomics workflowProper proteome analysis by mass spectrometry depends on upstream sample manipulations, including protein concentration and purification. Cleanup strategies typically rely on chromatography, not only for the relative high recovery and purification efficiency, but also for ease of use through automation. Unfortunately, with chromatographic approaches sample loss is an expected occurrence, especially for high molecular weight and/or hydrophobic (membrane) proteins. By contrast, organic solvent‐assisted protein precipitation is extremely effective at protein purification with minimal sample loss. Quantitative protein recovery is possible, though it is highly dependent on the pipetting skills of the individual. It is desired to create a robust protein precipitation protocol by automating the process, thereby maximizing recovery and purity on a high throughput scale. This presentation introduces a disposable two‐stage centrifugal cartridge, tailored to automate the precipitation process. The ProTrap XG comprises an upper filtration cartridge together with a removable reversed phase cartridge, attaching at its base. Protein samples are mixed with organic solvents in the upper filter chamber, which induces precipitation of proteins. The pellet is retained by the filter, while contaminants in the supernatant are discarded to waste through a brief spin in a desktop centrifuge. Multiple devices can be used simultaneously, improving throughput while maintaining consistency between samples. Following precipitation, protein pellets are re‐solubilized and subject to final purification through the reverse phase cartridge which is re‐attached to the base of the filter. The effectiveness of the ProTrap XG for recovery and purification of intact proteins in a top‐down proteomics workflow was discussed.Alan Doucette, A. Crowell and S. Rudolph, Department of Chemistry, Dalhousie University, Halifax, NS, CanadaAutomated SDS removal, tryptic digestion and sample cleanup in a disposable two‐stage spin cartridgePrior to MS analysis, bottom up proteomics relies on a significant number of sample manipulations including protein purification and preconcentration, fractionation, digestion, and subsequent cleanup. Each of these steps comes with a risk of loss of analyte and adds to the complexity of the proteomics workflow. The GELFrEE fractionation system is a mass‐based electrophoretic platform which relies on an SDS‐containing sample buffer to isolate protein fractions in solution. Our group has recommended the use of solvent‐induced protein precipitation (acetone or chloroform/methanol/water) to eliminate the SDS ahead of LC/MS analysis. However, protein precipitation is a particularly tedious and highly variable technique for the recoverery of proteins in high yield. Subsequent resolubilization of the protein pellet is also challenging, and further jeopardizes the success of MS characterization. To facilitate protein precipitation we have developed a two‐stage filtration and chromatographic extraction cartridge designed to work in a conventional benchtop centrifuge. The device automates the recovery of proteins, ensuring uniform purification on a high throughput scale. Protein samples are precipitated in an upper chamber, with residual contaminants (including SDS) flushed through the bottom filter. At this point the base filter is capped, trapping proteins in the upper vial for subsequent resolubilization. A combination of urea and trypsin ensures complete digestion of the pellet. The peptides are then subject to reversed phase purification; here we link a chromatographic cartridge to the base of the filter chamber. Key figures of merit of the device, including the purity of SDS removal, protein recovery, digestion efficiency and peptide recovery, were demonstrated through standard and complex proteome mixtures.Don Douglas1, C. Gao1, K. Kim1, H. Qiao2 and N. Konenkov31Department of Chemistry, University of British Columbia, Vancouver, Canada2Ionics Mass Spectrometry Group, Bolton, ON, Canada3Physical and Mathematical Department, Ryazan State University, Ryazan, RussiaSpace charge effects in linear quadrupole ion traps with mass selective axial ejection (Invited Talk)Experiments and ion trajectory calculations have been used to better understand and to reduce space charge effects in linear quadrupole ion traps operated with axial ejection. If too many ions are stored in a linear trap their mutual repulsion counteracts the trapping forces and the ion oscillation frequencies decrease. Consequently higher trapping radio frequency voltages are required to bring ions into resonance for ejection, and ions appear at higher apparent masses in a spectrum. At the same time the mass resolution decreases. For the experiments of interest here the mass shifts are about 1 at m/z 609, and the electric field from space charge is a small perturbation to the trapping field. Experiments show that a proper choice of operating conditions can reduce but not eliminate mass shifts. Trajectory calculations of ion motion with various models of the ion cloud provide additional insight, and show that the space charge field is nonlinear and that therefore the ion oscillation frequency depends on oscillation amplitude. Anything that expands the ion cloud produces lower electric fields from space charge, and hence lowers mass shifts. Experiments in progress show that using broadband excitation, dipole dc, or two‐frequency excitation to expand the ion cloud gives modest reductions in mass shifts and improvements in resolution.Norman Dovichi, Department of Chemistry and Biochemistry, University of Notre Dame, South Bend, Indiana, USACapillary electrophoresis for high throughput proteomics (Keynote Lecture)Most proteomics studies employ LC/ESI‐MS/MS analysis of peptides. We are investigating capillary electrophoresis (CE)/ESI‐MS/MS as an alternative technology for bottom‐up proteomics. We first developed a rugged and sensitive CE/ESI interface based on electrokinetically pumped sheath‐flow.[1] This interface operates in the nanospray domain, produces low‐attomole detection limits for CE separation of peptides, and offers great flexibility in separation buffer conditions.[1,2] We then analyzed the secreted protein fraction of M. marinum.[3] We pre‐fractionated the secretome to produce simpler samples that were better suited to the separation performance of capillary zone electrophoresis (CZE). The results of the analysis of 12 fractions were compared with conventional UPLC analysis of the unfractionated sample. CZE produced slightly more protein identifications in a slightly shorter time period than UPLC. 140 protein groups were identified by CZE/ESI‐MS/MS in three hours from this sample. We have recently improved the system. In the single‐shot analysis of the E coli proteome, we identified >1,300 peptides and >300 protein groups in a 50‐min CZE separation.[4] We have employed this separation system to analyze seven fractions from the E. coli proteome.[5] This system produced 23,706 peptide spectra matches, 4,902 peptide IDs, and 871 protein group IDs in 350 min analysis time. In an alternative separations scheme, we employed capillary isoelectric focusing for the analysis of host‐cell proteins in commercial biopharma products; this system identified 37 host cell proteins in a total sample preparation time of 4 hours.[6][1] Wojcik et al. Rapid Communun. Mass Spectrom. 2010, 24, 2554.[2] Wojcik et al. Talanta2012, 88, 324.[3] Li et al. Anal. Chem. 2012, 84, 1616.[4] Zhu et al. Anal. Chem. 2013, 85, 2569.[5] Yan et al. In revision[6] Zhu et al. Talanta2012, 98, 253.Ann M. English, Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec, CanadaPlatform development to monitor age‐ and oxidant‐related protein post‐translational modifications: The case of copper‐zinc superoxide dismutase (Invited Talk)Non‐enzymatic protein post‐translational modifications (PTMs) increase with age and are frequently associated with age‐dependent diseases. For example, copper‐zinc superoxide dismutase (CuZnSOD) aggregation has been reported in patients with late onset neurodegenerative diseases such as sporadic or familial Amyotrophic Lateral Sclerosis (ALS) and Alzheimer's. Non‐enzymatic PTMs in CuZnSOD are linked to its aggregation and hence to the development of ALS and Alzheimer's. Yeast is a widely used model of cellular and organismal aging that can be readily exploited in platforms to monitor age‐related PTMs in a cellular environment. Furthermore, yeast and human CuZnSODs share high sequence homology but, since yeast ages much faster, we are exploring age‐related PTMs in the yeast enzyme. By gel filtration of cell lysates we isolated a high‐mass inactive form of CuZnSOD, and found by FT‐MS that it is oxidized at cysteine 146 (C146) to the sulfonic acid. Importantly, C146 is also found in the sulfonic acid form in CuZnSOD isolated from the brains of Alzheimer's patients[1] and the C146R mutation has been reported in familial ALS.[2] Notably, C146 forms a C146‐C55 disulfide bridge that is critical in stabilizing the native CuZnSOD homodimer. In high‐mass CuZnSOD, we additionally observe oxidation of histidine 71 (H71) and H120, which ligate the active‐site zinc and catalytic copper, respectively. We hypothesize that age‐induced oxidation of C146, H71, and H120 contributes to the development of ALS as well as Alzheimer's by releasing the redox‐active catalytic copper, which would probably increase oxidative stress and CuZnSOD aggregation. By monitoring the GFP (green fluorescent protein) fluorescence of CuZnSOD‐GFP from an isogenic yeast strain expressing this fusion protein, we noted that the cellular content of high‐mass CuZnSOD‐GFP increases ~10‐fold from day 3 to day 7. We are currently characterizing CuZnSOD and CuZnSOD‐GFP from yeast cells over several weeks to establish if aggregated CuZnSOD continues to accumulate in old cells. CuZnSOD activity also is known to be inhibited by inflammatory processes in the airway epithelium, and C146 oxidation was reported in CuZnSOD purified from the erythrocytes of asthmatic patients.[3] To mimic the inflammatory state of asthma we are examining modification of CuZnSOD after challenging yeast cells with H2O2. Our highly sensitive LC/MS platform that tracks protein modification as yeast ages is providing in‐depth information at the molecular level that is critical towards understanding the functional significance of PTMs in the cellular context.[1] Choi et al. J. Biol. Chem. 2005, 280, 11648.[2] Andersen et al. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 2003, 4, 62.[3] Ghosh et al. Antioxid Redox Signal2012. DOI: 10.1089/ars.2012.4566.Daniele Fabris, The RNA Institute, State University of New York, The University at Albany, Albany, NY, USAMS‐based strategies for the elucidation of nucleic acid–ligand interactions (Invited Talk)Ribozymes and riboswitches have keenly demonstrated that the function of nucleic acids is not always linked to the genetic information coded in their sequence, but can also depend on their 3D structure and ability to interact with a variety of species present in the cell. Owing to the versatility afforded by mass spectrometry (MS) in RNA analysis this platform is rapidly assuming a prominent role in the investigation of structure‐function relationships, which is realized by supporting the full characterization of natural and man‐made RNAs as well as the elucidation of specific interactions with cognate nucleic acids, proteins, metals, and small molecule ligands. The talk illustrated strategies for accessing functional information for nucleic acid complexes and discussed possible hurdles and experimental pitfalls. Examples were provided in which MS‐based techniques are employed to characterize relevant interactions that could represent new therapeutic targets. Other examples illustrated approaches for elucidating the effects of metals and common nucleic acid ligands on such interactions. The experimental design necessary to assess the strength of binding/inhibition in either comparative or absolute fashion was also discussed. The potential afforded by ion mobility spectrometry (IMS) techniques was evaluated in the investigation of possible conformational effects associated with ligand binding. Owing to the extremely low sample consumption and high speed of analysis, these capabilities will be expected to greatly increase the utilization of MS‐based approaches in drug discovery and therapeutics development based on specific nucleic acid interactions.Bella Groisman, J. Petschnigg and I. Stagljar, Department of Biochemistry, University of Toronto, Toronto, CanadaA novel mammalian split‐ubiquitin proteomics approach as a tool for functional investigation of signaling pathways in human cellsThe membrane yeast two‐hybrid system (MYTH) is a robust technique for the identification of protein partners of integral membrane proteins. However, its implementation for mammalian proteins is limited due to differences between mammalian and yeast cells. Here we present a novel split‐ubiquitin‐based mammalian membrane two‐hybrid (MaMTH) technology that enables investigation of protein‐protein interactions (PPIs) in human cells. Briefly, the specific interaction between membrane 'bait' and 'prey' proteins, coupled to ubiquitin halves, allows reconstitution of functional ubiquitin. Subsequently, deubiquitinating enzymes cleave off the transcription factor coupled to the bait, thus activating reporter gene transcription. Using the MaMTH technology we successfully confirmed known PPIs of membrane proteins from various cellular compartments. Here we focus on the ErbB members of receptor tyrosine kinases, mainly on the EGFR wild type (WT) versus the oncogenic kinase‐active L858R version – dynamic interactomes and functional analysis. We demonstrate that MaMTH allows sensitive detection of phospho‐dependent and drug‐inhibited interactions. We show that EGFR WT recruits Shc1 adaptor in serum‐dependent manner, while oncogenic L858R shows constitutively stimuli‐independent binding. Furthermore, consistent with clinical data EGFR (L858R) interaction with Shc1 was efficiently inhibited by the EGFR small molecule inhibitor erlotinib (tarceva), while the secondary L858RT790M resistant mutant was not affected by the drug. In addition, we created a dynamic interactome of EGFR WT versus L858R with 200 predicted interactors, data which is of high importance for the cancer signaling scientific community. In summary, here we presented a novel powerful proteomics technology, which we are planning to develop as tool for high‐throughput proteomic research as well as a drug screening platform.David Juncker, Department of Biomedical Engineering, McGill University, Montréal, CanadaAntibody colocalization microarray (ACM): A scalable cross‐reactivity free nano‐ELISA platform for proteomics studiesQuantification of proteins with high specificity in blood can be performed using sandwich immunossays (e.g. ELISA). Multiplex sandwich immunoassays (MSI) were developed to measure multiple proteins at once, but cross‐reactivity among reagents is known to limit accuracy and reproducibility. Here we explain the origin of the cross‐reactivity in MSI, experimentally show the consequences, and introduce a novel assay format called the antibody Colocalization Microarray (ACM) that eliminates it. In MSI a slide is patterned with an array of capture antibodies, incubated with a sample, followed by a mixture of detection antibodies. Here we analyze the combinatorial interactions between detection and capture antibodies and antigens, and find that it increases quadratically with the number of targets. In an array with 14 targets we found widespread cross‐reactivity. To overcome the cross‐reactivity we introduce the ACM where all detection antibodies are individually spotted with high accuracy onto their corresponding capture antibody spot. Each spot on the ACM replicates an ELISA but using only 1 nL of reagent, constituting a Nano‐ELISA. An ACM with 50 targets was shown experimentally to overcome cross‐reactivity and to rival the sensitivity of ELISA. The ACM was validated for biomarker studies by measuring proteins in the serum of breast cancer patients and controls, and a candidate biomarker panel was identified. It is currently being used to identify biomarkers for traumatic brain injury. As the number of targets on the ACM is increased easily, it may become a powerful tool for biomarker discovery and validation.John Klassen, Department of Chemistry, University of Alberta, Edmonton, CanadaNew MS tools for the discovery and characterization of protein‐carbohydrate interactions (Invited Talk)The direct electrospray ionization mass spectrometry (ESI‐MS) assay has emerged as a powerful tool for quantifying protein‐ligand interactions in solution. The assay is based on the direct detection and quantification of free and ligand‐bound protein ions by ESI‐MS for solutions of known initial concentrations of protein and ligand. A brief overview of the ESI‐MS assay was presented, along with recent methodological advances that overcome the major sources of error in the binding measurements. A high‐throughput ESI‐MS approach to library screening was also presented. The catch‐and‐release (CaR)‐ESI‐MS assay involves incubating a protein with a library of compounds in solution, detecting the protein‐ligand complexes by ESI‐MS, activating the complexes to release the ligand. The identities of ligands are determined from measured molecular weights and, if needed, the fragmentation spectra and ion mobility arrival time distributions. The CaR‐ESI‐MS assay allows for the sensitive, rapid (<1 min analysis time) and direct detection and quantification of specific interactions for libraries containing hundreds of compounds. An overview of the assay was presented followed by examples highlighting the application of the assay for the discovery of carbohydrate ligands for a variety of bacterial proteins. Finally, the extension of the CaR‐ESI‐MS assay for the discovery of host cell receptors was described. The assay involves direct ESI‐MS analysis of aqueous solutions of soluble proteins and insoluble receptors, which are incorporated into nanodiscs. The application of this assay for the detection of interactions between bacterial toxins and glycosphingolipids was presented.X. Chris Le, H. Zhang, F. Li, B. Dever, X. Li, C. Wang, M. Wagner, Z. Wang and X.‐F. Li, Departments of Chemistry and of Laboratory Medicine and Pathology, University of Alberta, Edmonton, CanadaDNA‐mediated binding assays for nucleic acids and proteins (Invited Talk)The detection of nucleic acids and proteins is fundamental for studying their functions and for the development of molecular diagnostics. Determining these biomolecules in complex systems requires exquisite analytical specificity and sensitivity. To meet these requirements we have devoted much effort to affinity binding assays that incorporate target recognition, signal transduction, and in situ amplification. Here we focus on homogeneous binding assays, which are carried out in solution, without the need for separation, immobilization, or washing steps. Such homogeneous binding assays can be performed in a single tube/vial or in live cells. They are potentially applicable to point‐of‐care diagnostics. For example, a binding‐induced DNA assembly enables ultrasensitive detection of molecular targets and construction of unique target‐dependent nanoreactors. Two DNA motifs that are conjugated to specific affinity probes assemble preferentially only when a specific target triggers a binding event. The binding‐induced assembly of the DNA motifs results in the formation of a highly stable hairpin structure, enabling effective differentiation of the target‐specific assembly from the background. This strategy pushes the limit of the state‐of‐art dynamic DNA nanotechnology to include molecules beyond DNA. The ability to generate dynamic DNA assembly by non‐DNA molecules opens up opportunities for diverse potential applications, ranging from the construction of protein‐induced nanodevices to the in situ detection of proteins and studies of molecular interactions.XingFang Li, W. Wang, Y. Quian and J. M. Boyd, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada.Mass spectrometry methods for characterization and determination of disinfection byproducts in tap water and swimming poolsProtection of public health requires disinfection of drinking water and recreation pools to effectively prevent water‐borne diseases. However, common disinfection treatments unavoidably generate byproducts resulting from reactions between natural organic matter in the source water and disinfectants. The majority (~70%) of disinfection byproducts (DBPs) in treated water remain unidentified. Potential adverse health effects observed in epidemiological studies drive analytical research to identify and determine what DBPs may contribute to these health risks. Here we present the separation of and mass spectrometry methods to characterize emerging DBPs of toxicological relevance, and investigate the occurrence of these DBPs in swimming pools. The research demonstrated that personal care products contributed to the formation of some emerging DBPs such as halobenzoquinones.Yueqiao Fu, Lily Mats, Graham T. T. Gibson and Richard Oleschuk, Department of Chemistry, Queens University, Kingston, Ontario, CanadaMulti‐nano ESI, in pursuit of enhanced robustness, stability and sensitivity (Invited Talk)Electrospray ionization (ESI) as a soft ionization technique has been critical to mass spectrometry for analyzing biological macromolecules. NanoESI, which is operated at lower flow rates (nL/min), exhibits both higher ionization efficiency and higher sensitivity since the smaller initial electrospray droplets from the emitter afford increased surface charge per analyte molecule. To improve sensitivity and signal without decreasing flow rate, multiple electrosprays have been generated from multiple emitters by splitting one larger flow into smaller flows in the nanoESI regime. Based upon theoretical prediction and experimental results, the measured spray current generated from an emitter producing multiple Taylor cones (Itotal) is enhanced relative to the spray current from a single emitter (Is) at the same overall flow rate by a factor of the square root of the number of emitters (n). We have developed a series of multi‐channeled emitters based upon silica and polymer microstructured fibers (MSFs). Custom‐designed MSFs having holes in a radial pattern were fabricated using a 'stack and draw' technique, allowing multiple electrosprays from a single fiber. Polymer nozzles have been included in the silica MSF design to enhance nanoESI performance. A layer of polymer is first formed on the inner wall of each of the MSF channels followed by etching with hydrofluoric acid to expose the nozzles. The protruding highly cross‐linked divinylbenzene nozzles produce true multi‐nanoESI over a wide range of flow rates and mobile phase compositions and are compatible with high‐resolution liquid chromatographic separations.Anthony Pawson, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, CanadaUsing synthetic biology to study protein signaling networks (Plenary Lecture)The proteins involved in the transmission of signals within cells are typically complex, in the sense that they often possess multiple interaction and/or catalytic domains, and are directly or indirectly connected to numerous distinct proteins and other biomolecules such as phospholipids. We have used a variety of synthetic biology and proteomics techniques to investigate the importance of the multiple protein‐ and lipid‐binding domains of the Grb2 adaptor protein and its target, the Ras guanine nucleotide exchange factor Sos1, in controlling a key cell fate decision in early mammalian embryogenesis, namely the differentiation of primitive endoderm tissue. We find that these domains act cooperatively as a coincidence detector to ensure that fibroblast growth factor‐dependent signals are transmitted at the right time and in the right place to elicit the appropriate formation of primitive endoderm.Guy Poirier, J.‐P. Gagné, J. D. Chapman and D. R. Goodlett, Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University, Québec, PQ, CanadaHigh accuracy mass spectrometry provides direct evidence for site‐specific auto‐ADP‐ribosylation of PARP‐1Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is an abundant chromatin‐associated protein involved in a variety of pathways such as DNA damage response (DDR), transcription regulation, cell cycle progression and apoptotic cell death. Upon DNA damage induction, DNA‐dependent PARPs synthesize an anionic poly(ADP‐ribose) (pADPr) polymer to which several proteins bind with the subsequent formation of pADPr‐associated multiprotein complexes. PARP hydrolyzes NAD + in the formation of pADPr on nuclear proteins such as DNA repair enzymes, chromatin remodelers, histones, transcription factors and PARP‐1 itself. The covalent attachment of pADPr to a substrate protein alters its physicochemical characteristics, which results in functional consequences on its biological activity. The exact nature of the ADP‐ribose linkage catalyzed by PARP‐1 has been the subject of controversy and debate. To date, it is still unclear which of the amino acid side chains are directly targeted by the ADP‐ribose transferase activity of PARP‐1. Radiolabeled NAD + has been a cornerstone in research for tracking and visualizing PARP‐catalyzed post‐translational protein modification in vitro, but the ability of the negatively charged pADPr to strongly interact with proteins in a non‐covalent manner is a cause of confusion when interpreting experimental results. To truly begin to understand the exact nature of the poly(ADP‐ribosly)ation reaction, there is a need for more conclusive mass spectrometry evidence of PARP‐catalyzed protein modifications. Mapping of these modifications to specific amino acid residues on PARP‐1 and other protein substrates of poly(ADP‐ribosyl)ation reactions presents significant challenges due to the complex structural heterogeneity of pADPr polymers as well as a loss of amino acid sequencing information that occurs during several mass spectrometry peptide activation techniques commonly implemented for fragmentation. Here, we demonstrate a novel method for the identification of PARP‐1 auto‐poly(ADP‐ribosyl)ation sites utilizing high mass accuracy mass spectrometry. By employing an enzymatically driven simplification of the pADPr modifications, we reduce the corresponding complexity of analyzing modified tryptic peptides at the level of the tandem mass spectrum, which in turn enables us to effectively localize exact sites of PARP‐1‐catalyzed poly(ADP‐ribosyl)ation. In conjunction with a phosphopeptide‐like enrichment strategy that captures modified peptides, we are able to successfully locate numerous sites of PARP‐1 automodification in a high‐throughput proteomics screen suitable for universally available mass spectrometry and data analysis pipelines.Jeffrey Smith, K. Wasslen, S. Wood and J. Manthorpe, Department of Chemistry, Carleton University, Ottawa, CanadaTrimethylation enhancement using diazomethane (TrEnDi): Rapid on‐column methylation of peptides and proteins to permit quantitative analysis using tandem mass spectrometryTrimethylation enhancement using diazomethane (TrEnDi) is an inexpensive and rapid on‐column chemical derivatization technique to permit peptide methylation and quantitation. TrEnDi produces trimethylated primary amines that enhance and simplify label‐free quantitation strategies via improved ionization through the formation of fixed quaternary ammonium ions. TrEnDi has been optimized on a microfluidic platform to successfully derivatize peptides from a protein digest with nearly 100% complete N‐terminal methylation. Other amino acid side chains with pKa values of approximately 10.5 or less were also methylated. MS/MS analysis confirmed that N‐termini trimethylated peptides fragmented to preferentially form the a2 ion, permitting a significant increase in sensitivity when scanning in the MRM mode. This allowed us to perform label‐free MRM scanning of candidate peptides without prior knowledge of their fragmentation patterns. The trimethylated peptide N‐termini were found to be very stable. However, hydrolysis of the newly formed methyl esters occurred over time. Hydrolysis was assisted using base hydrolysis in order to drive the derivatized peptides to homogeneity. To test the analytical sensitivity of derivatized peptides via MRM scanning, several low concentrations of the modified peptides were spiked into a series of more complex peptide solutions and the ability of the MRM method to obtain quantitative data was assessed. Our results have revealed that the trimethylated peptide N‐termini produced strong MRM transitions and were identifiable at low concentrations, even in the presence of highly concentrated interferences.E. Bonneil1, F. Lamoliatte1,2, J. Saba4, O. Caron‐Lizotte1 and Dr P. Thibault1,2,3, Institute for Research in Immunology and Cancer1, and Departments of Chemistry2 and of Biochemistry3, Université de Montréal, Montréal, Canada, and Thermo Fisher Scientific4Quantitative proteomics using FAIMS and isotopic labeling (Invited Talk)Isotopic labeling is a commonly used method in quantitative proteomics to profile changes in protein abundances across different cell conditions. Isotopic labeling can be performed using chemical reagents that target specific functional group on peptides (MS2 quantitation) or via metabolic labeling (stable isotope labeling of cell cultures, SILAC) where cells are cultured in media containing in either light or heavy isotopes of amino acids (MS quantitation). The predictable mass differences between peptides (or fragment ions) from two or more experimental conditions enable a direct quantification and provide precise functional information and temporal changes in the proteome. However, the dynamic range and precision of these quantitative measurements can be hampered by the increasing complexity of the mass spectra and the presence of co‐eluting isobaric ions. In this context we evaluated the analytical potentials of high field asymmetric waveform ion mobility spectrometry (FAIMS) in large‐scale quantitative proteomics experiments using stable isotope labeling on a LTQ‐Orbitrap Elite mass spectrometer. The ability of FAIMS to separate multiply protonated compounds from singly charged background ions provided a unique advantage to extend the dynamic range of peptide detection while reducing the complexity of LC/MS peptide maps. FAIMS analyses performed on cell extracts derivatized with tandem mass tags (TMT)‐6plex isotopic label reagent or grown under SILAC conditions provided improved quantitation and dynamic range compared with conventional nanoelectrospray. Quantitative proteomics analyses performed using FAIMS resulted in a reduction of mixed precursor ions and provided confident reporter ion measurements with increased peptide identification. Examples of application were presented for monitoring changes in the proteome of yeast and human cells.Ruijun Tian, Y. Shi, T. Hunter and T. Pawson, The Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, CanadaExploring bi‐directional signaling in pancreatic tumor microenvironment by quantitative proteomicsPancreatic ductal adenocarcinoma (PDA) is one of the most lethal human cancers with resistance to many chemotherapeutic drugs. This drug resistance effect is believed to be partly caused by stromal‐cancer cells interaction in pancreatic tumor microenvironment. Cell‐cell interactions are often mediated by protein molecules expressed by one cell that are recognized by specific receptors present on neighboring cells. This intercellular communication activates specific signaling pathways through the induction of dynamic post‐translational modifications such as phosphorylation and by protein‐protein interactions. Here, we present the development of unbiased quantitative proteomics approaches to characterize a dynamic secretome and critical intracellular signaling in a simulated pancreatic tumor microenviroment in culture. A number of secreted ligand/receptor pairs have been explored which have significant effects on cancer cells proliferation. Our work establishes a generally applicable strategy for studying stromal‐cancer cells communication and provides valuable resources for systematically understanding dysfunctional signaling networks in tumor microenvironment in a context closer to physiological conditions than was previously possible.Sarah Trimpin, Department of Chemistry, Wayne State University, Detroit, MI, USAMatrix assisted ionization without a laser, heat, or voltage: identification of peptides and proteins directly from tissue (Invited Talk)A few years ago we introduced an ionization method for use in mass spectrometry (MS), applicable to volatile and nonvolatile compounds, which uses laser ablation of a matrix/analyte mixture similar to atmospheric pressure matrix‐assisted laser desorption/ionization (MALDI) but which produces mass spectra nearly identical to electrospray ionization (ESI). This new technique called laserspray ionization (LSI) has advantages of speed of analysis, high spatial resolution for imaging, mass range extension, and improved fragmentation common with multiply charged ions. LSI using the proper matrix was also shown to be capable of imaging multiply charged ions using atmospheric pressure or vacuum MALDI sources. Crucial for the production of highly charged ions are desolvation conditions to remove matrix from charged matrix/analyte clusters. By using a matrix that readily sublimes, we have now extended matrix‐assisted ionization by eliminating the need for a laser or even heat to initiate ionization. Ions are spontaneously formed from a solid matrix/analyte material when introduced to a vacuum MALDI source without need of a laser, or to a modified ESI source without use of voltage. This matrix assisted ionization vacuum (MAIV) method produces similar charge states and drift times of ions, as determined by ion mobility spectrometry‐MS, to ESI. Compounds at least as large as bovine serum albumin (67 kDa) become converted into highly charged gas‐phase ions when exposed in the MAIV matrix to vacuum conditions. Such a simple ionization method, requiring only the vacuum necessary for the proper functioning of the mass spectrometer, may prove useful for analysis of single cells and in clinical applications. A possible mechanism for ionization involving sublimation/evaporation and charge generation through the mechanism that produces triboluminescence was discussed.Dietrich Volmer, Institute of Bioanalytical Chemistry, Saarland University, Saarbrücken, GermanyNew methods for improved determination of metabolic markers using advanced analytical mass spectrometry techniques (Invited Talk)New developments in metabolomics and metabolic profiling techniques have led to the discovery of metabolic biomarkers indicative of physiological or pathological change, e.g. onset of disease or nutritional effects of a specific diet. Experimental approaches for discovering new biomarkers usually involve proteomics or metabolomics techniques that detect differences between sample sets rather than measuring specific compounds as performed in traditional hypothesis‐driven research. Unfortunately, finding metabolites present in significantly different levels between sample sets is often complicated by experimental variables such as method reproducibility, types of tissue sampling technique, analytical sample preparation technique, ion suppression phenomena, person‐to‐person or animal‐to‐animal metabolic variations, isobaric or isomeric noise, etc. All these error sources, variations or artefacts can lead to false assignment of metabolites or biomarkers and random and systematic errors during quantification. This presentation focused on our research on metabolite profiling techniques and sources of analytical errors. It presented examples for applications such as quantitative analysis and fingerprinting of vitamin D metabolites, microcystin toxin biomarkers and lipids. In addition, novel technical solutions involving “magnetic” media (fluids and beads), ion mobility spectrometry and chemical labeling techniques were highlighted in this presentation.Fangjun Wang1, H. Zou1, M. Ye1 and Daniel Figeys21Key Lab of Separation Science for Analytical Chemistry, National Chromatography R&A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Science, Dalian, China;2Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Canada.Recent development of technologies and methods for proteome analysis (Invited Talk)Technologies and methods play a highly important role for proteome analysis. Recent developments of technologies and methods for proteome analyses in our labs were presented:Combined technologies and methods, such as multi‐enzyme digestion, different enrichment methods, multi‐dimensional LC separation and different types of mass spectrometers, for comprehensive glycoproteome and phosphoproteome analysis of human liver; the largest dataset of protein phosphorylation and glycosylation for human liver was achieved.Solid‐phase based technology by integrating all of the digestion, enrichment, and deglycosylation together with LC/MS analysis, was developed for glycoproteome analysis; both the identification sensitivity and the throughput were improved greatly.A solid‐phase based labeling approach, by integration of glycopeptides and phosphopeptides enrichment and stable isotope labeling on adsorbent beads, was developed for relative quantification of protein glycosylation and phosphorylation.Six‐plex stable isotope labeling was achieved by a two‐stage stable isotope labeling strategy of SILAC and dimethylation isotope labeling, which allows six different protein samples (six‐plex) to be reliably labeled and simultaneously quantified at MS1 level. This six‐plex isotope labeling strategy was applied to investigate the dynamics of protein turnover, and the 50% turnover times of 1365 proteins within HeLa cells were successfully obtained.Hailin Wang, S. Liu, B. Zhao, D. Zhang, C. Li, State Key Laboratory of Environmental Chemistry and Ecotoxicology, Chinese Academy of Sciences, Beijing, China.Insights on Isotachophoresis Kinetics from Monitoring Non‐uniform Motion of single DNA molecules (Invited Talk)Isotachophoresis (ITP) has wide applications in chemistry and life sciences due to its pre‐concentration function. A simplified ITP system consists of a leading electrolyte (LE) and a terminating electrolyte (TE). For LE and TE to migrate at the same velocity (v), the respective E in each zone must adapt to their electrophoretic mobility (μ) according to Eqn. (1): urn:x-wiley:09514198:media:rcm6724:rcm6724-math-0001The LE zone with a high electrophoretic mobility will have a low E, and the TE zone with a low electrophoretic mobility will have a high E. Current literature commonly assumes that the adaption of E is fast and is not the rate‐limiting step. However, actual adaption kinetics of ITP has not been studied yet. Moreover, it is not clear how electroosmotic flow affects the kinetics of ITP. By taking advantage of single molecule imaging, we for the first time examined the changes in electric field and the resulting non‐uniform motion of single DNA molecules in capillary isotachophoresis. The individual DNA molecules passing the detection window are consecutively imaged in real time at 50‐millisecond intervals. Since the migration velocity of DNA is directly proportional to the applied electric field strength (E), imaging the movement of DNA provides necessary information for understanding the distribution of E throughout the capillary. This approach allows us to gain insights into the kinetics of varying‐field ITP and enables us to develop a strategy for focusing and detecting single DNA molecules and DNA damages.John Wilkins, Departments of Internal Medicine, Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, CanadaLinking protein composition and functionality in biology and medicine (Invited Talk)The repertoire and functional state of proteins expressed by an organism ultimately determine their phenotype. Thus, the inclusion of measures of protein activity is an essential step to developing valid models of biological systems in disease and health. While genomics and proteomics provide detailed information on potential and current protein composition, these approaches generally offer limited insight regarding protein functional states. However, many proteins possess catalytic activities that are stringently controlled by mechanisms that restrict substrate accessibility to the active site. This is the central premise of activity‐based protein profiling. The use of enzyme activity as a predictor of organ function or health offers several advantages in the clinical environment. The assays can be highly specific because of the structural features of their substrates. The high substrate turnover numbers of enzymes provides an amplification mechanism that increases assay sensitivity. Activity‐based profiling offers a means of probing clinical samples for novel enzymatic activities as new reporters. Some of our experiences in the application of this approach profiling assessing renal function were presented.The identification of a protein, even an active one, does not necessarily address the questions of “What does the protein do?” and “Why is it needed?”. These questions can be further complicated by the observations that function(s) are often context specific. We have used integrated proteomic, activity based protein profiling and functional genomics approaches in an effort to examine lymphocyte migration, a process that is central to normal immune function.Derek Wilson, T. Rob, D. Golemi‐Kotra and P. Kama‐Gill, Department of Chemistry, York University, Toronto, CanadaMicrofluidics‐enabled, sub‐second hydrogen/deuterium exchange pulse labeling reveals allosteric 'hotspots' in enzymesRegulation of metabolic pathways is achieved by modulation of enzyme activity, commonly via direct interactions at the active site. However, a substantial number of enzymes are also sensitive to changes at locations well removed from the site of catalysis. These 'allosteric' effects are easily detected, but are exceedingly difficult to characterize because they are usually driven by subtle changes in conformation or dynamics. In this work we use a new microfluidic chip that incorporates a bottom‐up workflow with sub‐second H/D exchange pulse labeling to probe time‐dependent structural and dynamic changes in the beta‐lactamase enzyme TEM‐1 upon inhibitor binding. Changes in deuterium uptake were noted from the first time‐point after acylation, corresponding to both increases and decreases in labeling ('loosening' and 'tightening' of the structure, respectively). As the acyl‐enzyme was allowed to age, time‐dependent changes in deuterium uptake were detected, indicating the presence of slow structural changes occurring up to 2 s after acylation. In general, peptides showing instantaneous changes in uptake were located near the active site, while peptides that exhibited time‐dependent changes in deuterium uptake were located at the periphery of the enzyme. In the active site, patterns of increasing and decreasing rigidity explain the roles of Arg244 and Asn276 in the inhibitory mechanism. At the periphery, slow changes in uptake are observed specifically in allosteric 'hotspots' that include residues known from mutational analysis to play a role in TEM‐1 catalytic efficiency.David Wishart, Departments of Biological Sciences and Computing Science, University of Alberta, Edmonton, CanadaClearing the biomarker barrier: Moving biomarkers from the bench to the bedside (Invited Talk)One of the great promises of the 'omics' era was the delivery of a gene or a protein to diagnose every disease. Unfortunately this hasn't quite happened. While our understanding of the etiology of disease has improved tremendously our ability to diagnose or predict disease has not. This has a lot to do with our inability to translate biomarkers from the bench to the bedside. Indeed, after nearly 20 years of work and more than $5 billion in investment, there have been only a few microarray tests and, as yet, no protein‐array or proteomic tests approved by the FDA for diseases diagnosis. On the other hand, dozens of metabolomic biomarkers have successfully made the transition from the lab to the clinic. Why? This presentation discussed some of the secrets to the success of metabolomics in clearing the 'biomarker barrier'. In particular, some key guidelines for designing, performing and reporting biomarkers in a biomarker study, as well as some advice with regard to the process of moving biomarkers from the laboratory to the clinic, were provided. Some examples of how quantitative metabolomics is being used to identify biomarkers for a wide range of diseases, and the status of my lab's efforts to try to move some these biomarkers into clinical testing, were also discussed.THE KEN STANDING AWARD LECTURE (Sponsored by the University of Manitoba)Lars Konermann, Departments of Chemistry and of Biochemistry, The University of Western Ontario, London, ON, CanadaElectrospray mass spectrometry as a readout of protein structure and functionProteins are nanomachines that carry out countless tasks in every living organism. To perform these functions, the linear amino acid chain of each protein has to fold into a highly specific three‐dimensional structure. On the other hand, misfolding and aggregation are associated with diseases such as Alzheimer's and Parkinson's. X‐ray crystallography remains the gold standard for obtaining high‐resolution structural information. However, the data generated in this way do not properly reflect the highly dynamic nature of proteins, which is a key prerequisite for biological function. Electrospray ionization mass spectrometry (ESI‐MS) provides a number of avenues for exploring protein structure, function, folding, and dynamics. Our laboratory specializes in the use of hydrogen exchange and covalent labeling techniques. This presentation discussed time‐resolved investigations that provide detailed insights into the mechanisms of protein folding. The area of membrane protein structure and function, and the use of ESI‐MS for probing biological self‐assembly processes, were also discussed. In addition, recent advances in understanding the physical processes that allow the formation of gaseous biomolecular ions during ESI were presented.THE BILL DAVIDSON GRADUATE STUDENT TRAVEL AWARD (Sponsored by ABSciex)Feng Li, Department of Chemistry, University of Alberta, Edmonton, CanadaDr X. Chris Le, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, CanadaDynamic DNA assemblies mediated by binding‐induced DNA strand displacementDynamic DNA assemblies have shown promising potential to regulate cell activities, deliver therapeutic compounds, and amplify detection signals for molecular diagnostics and imaging. However, such applications of dynamic DNA assembly systems have been limited to nucleic acids and a few small molecules. It is challenging to construct dynamic DNA assemblies that are responsive to proteins. We have developed a binding‐induced DNA strand displacement strategy that can convert protein binding to the release of a predesigned output DNA at room temperature with high conversion efficiency and low background. This strategy allows us to construct dynamic DNA assembly systems that are able to respond to specific protein binding, opening an opportunity to expand the existing dynamic DNA nanotechnology to proteins for diverse applications. We have applied this strategy to design a binding‐induced DNA strand displacement beacon and a binding‐induced DNA circuit, both of which are triggered by protein binding. We have demonstrated the detection of platelet‐derived growth factor (PDGF) and prostate specific antigen (PSA). Further applications could be in the area of point‐of‐care analysis of proteins that could be performed under ambient temperature and without requiring the use of enzymes for signal generation and/or amplification.POSTER PRESENTATIONSJ. Anichina, A. Schreiber, R. Bonner and T. SakumaHigh‐resolution tandem mass spectrometry analysis of the interactions of oligonucleotides with selected river basin specific pollutantsE. Forsberg and J. BrennanBio‐solid phase extraction and information dependent tandem mass spectrometry for the identification of enzyme inhibitors in complex mixturesH. Li, S. Bergeron, and D. JunckerSnap chip 2.0: Double microarray‐to‐microarray transfer for easy‐to‐use, high‐density antibody colocalization microarraysP. Liuni and D. J. WilsonInvestigating catalysis‐linked dynamics in yeast alcohol dehydrogenase by measuring kinetic isotope effects using time‐resolved ESI‐MS with H/D exchange.P. S. Lo, V. Laforte, S. Bergeron, J. Marcoux and D. JunckerUsing the antibody colocalization microarray to measure proteins in complex samples from severe traumatic brain injury patientsZ. Ning, D. Seebun, B. Hawley, C.‐K. Chiang and D. FigeysFrom cells to peptides: 'One‐stop' integrated proteomic processing using AmphipolsD.J. Orton, J.P. Rogers and A.A. DoucetteA dual capillary nanospray interface for improved throughput in LC/MSA. SardariTRAQ coupled 2D‐LC‐MALDI‐TOF/TOF analysis reveals proteome changes associated Leishmania donovani promastigote adaptation to oxidative and nitrosative stressA. M. Smith and J.D. BrennanDevelopment of a multiplexed kinase assay via MALDI‐MS/MS detectionJ. Song, X. Tang, L. Li, L.C. Palmer, D. Pinto and K. ChisholmOFFGEL peptide pre‐fractionation is essential for proteomic approaches employing quantitative stable isotope dimethyl labeling and multiple reaction monitoring (MRM) in fruit proteomic researchC. StephanPushing the frontier in ICP‐MS applications: Counting and sizing nanoparticles using single particle ICP‐MSD. Williams, S. Madler, L.V. DeSouza, A. Matta and K.W.M. SiuIntracellular signalling network expression profiling by LC/MS with a heavy isotope‐labelled QconCAT internal standardInformation about past and future symposia and conferences organized by ETP can be found at the website www.etpsymposium.org.Robert K. Boyd, Researcher Emeritus, National Research Council of CanadaChair, Science Advisory Committee, ETP