SYMPOSIUM CHAIRS:
Christoph Borchers, University of Victoria
K.W. Michael Siu, York University
SPEAKERS
Ruedi Aebersold, Institute for Molecular Systems Biology, ETH Zurich
Mapping and Measuring Proteomes
The human genome project has taught us that a complete map -in the case of the genome project the complete genomic sequence – along with computational tools to navigate the map - represent invaluable resources for experimental and theoretical biologists. A main consequence of such a complete map is that all the biological processes have to be explainable with the components that constitute the map. Proteomics has not reached the stage that complete maps are available but the urgent need for their generation is now widely recognized.
In this presentation we will discuss experimental and computational challenges related to the generation of complete proteomic maps. We will also discuss recent technical advances towards complete proteome analysis and describe software tools and data resources that will transform proteomics from perpetual proteome mapping to accurate proteome measurement.
Alexandre M.J.J Bonvin, Utrecht University
Information-driven modelling of biomolecular complexes
With the presently available amount of genetic information, a lot of attention focuses on systems biology and in particular on biomolecular interactions. Considering the huge number of such interactions, and their often weak and transient nature, conventional experimental methods such as X-ray crystallography and NMR spectroscopy will not be sufficient to gain structural insight into those. A wealth of biochemical and/or biophysical data can however easily be obtained for biomolecular complexes. Combining these data with docking, the process of modeling the 3D structure of a complex from its known constituents, should provide valuable structural information and complement the classical structural methods.
We have developed for this purpose a data-driven docking approach called HADDOCK (High Ambiguity Driven protein–protein DOCKing) (http://www.nmr.chem.uu.nl/haddock) which is now also available as web server (http://www.haddocking.org/). HADDOCK distinguishes itself from ab-initio docking methods in the fact that it encodes information from identified or predicted protein interfaces in ambiguous interaction restraints (AIRs) to drive the docking process. Flexibility is accounted for in different ways during the docking which allows to model (small) conformational changes taking place during complex formation.
In my talk I will discuss the various sources of data that can be used to map interactions and illustrate their use in HADDOCK with examples from our laboratory together with results from our participation to the blind docking experiment CAPRI (Critical Assessment of PRedicted Interactions) (http://capri.ebi.ac.uk).
Joshua J. Coon, University of Wisconsin - Madison
How hybrid mass spectrometers with multiple analyzers and dissociation methods will transform protein sequence analysis
We describe the use of new mass spectrometry technology – an ETD-enabled Orbitrap – to characterize and quantify proteomes. The new instrument allows for the implementation of multiple dissociation methods, i.e., ion trap CAD, beam-type CAD (HCD), and ETD, and for the automated selection of each in a real-time based on multiple precursor attributes (i.e., data-dependent decision tree). Protein quantification is readily accomplished through use of isotopic labels – either SILAC or iTRAQ. The instrument will likewise propel top-down proteomics as acquisition of ETD-MS/MS spectra in the high resolving power Orbitrap allows for direct analysis of intact proteins on a sub-second timescale with ~ 300 ppb mass accuracies. Such mass accuracies are used to directly annotate ETD tandem mass spectral peaks with ion type and chemical composition. We demonstrate these and many other aspects of the instrument on a variety of applications involving human ES cells, differentiating human ES cells, and induced-pluripotent cells.
Catherine E. Costello, Boston University School of Medicine
Characterization of macromolecular assemblies and post-translational modifications associated with protein-based diseases
It is now possible to directly document differential protein expression and the post-translational modifications (PTMs) of proteins that arise via multiple mechanisms and to correlate these with the expressed phenotypes. We undertake development of MS-based technologies and methodologies for the identification, characterization and quantitation of known and novel PTMs, with emphasis on protein glycosylation and PTMs in pathways associated with oxidative stress, cardiovascular disease, and disorders with aberrant protein folding and/or glycosylation. We utilize microorganisms, animal models and clinical samples, and employ detailed protocols which incorporate abundant protein depletion, robust and novel multi-dimensional protein separations, high performance MS and tandem MS, and AFM imaging of fibrillar and aggregate protein deposits, followed by customized data analysis. Correlation of proteomic, glycomic, AFM and genomics data should lead to a better understanding of the clinical manifestations of disease states and physiological responses. These approaches bring with them both amazing opportunities and serious responsibilities: they hold great promise for increasing adoption into patient care and thus the training of present and future members of the mass spectrometry community will need to be attuned to the special requirements for clinical applications.
Norm Dovichi, University of Washington
Ultrasensitive proteomics
"The coupling of capillary electrophoresis with laser-induced fluorescence detection has resulted in a tool with unprecedented performance in bioanalysis. Capillary electrophoresis routinely produces separation efficiency over 1,000,000 theoretical plates. By coupling different forms of electrophoresis, two-dimensional capillary electrophoresis produces separations with over 1,000 resolution elements. Laser-induced fluorescence produces detection limits in the zepto- to yoctomole range, with single molecule detection possible in favorable cases. The technology played a key role in the sequencing of the human genome, and is now being developed for chemical cytometry, which is the analysis of the composition of single cells."
Andrew Emili, University of Toronto
Genetical and Chemical Proteomics
The sequencing of the human genome has turned our attention to the functions of the protein end-products. We want to know how proteins work together to make a cell, how they endow cells with the ability to respond to signals and organize into multi-cellular tissues of remarkable complexity, and how aberrations in protein expression or function lead to diseases such as cancer. Geneticists have traditionally approached these problems by trying to identify sets of genes that influence a trait in question. Ten years ago, microarray technology opened a second front by making it possible to compare expression levels for genes active under a variety of physiologic, environmental or pathologic conditions. A typical study reveals up- or down-regulation of pathways associated with a phenotype (case/control) or condition (treated/untreated). Likewise, chemical genomics offers new ways to dissect protein function by producing probes that can selectively modulate protein function. In the past few years, several groups have started to combine proteomic studies with genetic and chemical-genomic procedures, with the aim of creating a new synergy between these approaches. Here, I explore methods that seek to interface protein mass spectrometry with the analysis of genetic variants, perturbagens and quantitative phenotypes to systematically explore biological systems with enhanced resolution.
Gerard Hopfgartner, Universite de Geneve
Application of mass spectrometry for the absolute quantitation of proteins in biological matrices
Liquid
chromatography coupled to tandem mass spectrometry
(LC-MS/MS) has become a very promising analytical tool for
the absolute quantitation of therapeutic or endogenous
proteins in biological matrices such as plasma. All
approaches for the absolute quantitative analysis of
proteins include the following steps: 1) selective sample
preparation 2) digestion of the protein 3) quantitation of
signature peptides, as surrogates of the protein, on a
triple quadrupole using the multiple reaction monitoring
mode (LC-MRM/MS). Ideally, one would like to quantify a
large number of proteins with good precision and accuracy
as sensitive as possible, with a large dynamic range and in
a high-throughput fashion. Analytically, the procedure
seems to be quite straight forward but due to the
complexity of plasma the performance of the assay can be
jeopardized by many factors. A key challenge is the lack of
analytical selectivity of the signature peptide used for
quantitation versus other peptides from the biological
matrix. One way to improve the selectivity is to apply
column-switching approaches but to maintain high-throughput
the cycle time has to be as low as 5 minutes. Another
strategy is based on the use of triple quadrupole linear
ion trap where it is possible to combine MRM and
MS3
to
improve the selectivity of the signature peptide
quantitation. Finally, the potential of MALDI-MRM for ultra
fast quantitation of peptides will also be
discussed.
Juergen Kast,
University of British Columbia
Utilization
of proteomic database information in the study of human
blood platelets
Improvements
in mass spectrometric instrumentation continue to increase
the speed and sensitivity of peptide sequencing. This
enables the collection of information on more than a
thousand proteins in each sample within a few days. This
results in a depth of proteome coverage that makes
“proteomics”, the comprehensive analysis of the protein
complement of a cell or tissue, an attainable goal.
Moreover, it also accelerates the acquisition of such large
data sets and increases throughput. With more and more
large-scale proteomic studies being carried out, their
results will increasingly populate data repositories,
making them a valuable resource of information. Such
databases may be utilized in different ways. They can be
used post acquisition as a reference, against which new
data sets can be compared to determine data quality and
consistency, to reveal hidden patterns that are not
apparent from a single data set, but also to highlight
differences between experiments and samples. Alternatively,
repositories could also be interrogated prior to sample
analysis, such that they guide the experimental design,
allowing the prediction of expected experimental patterns
or selection or validation of selected peptides. Finally,
they could be employed to query data sets for features that
are independent of the actual experiments in which they
were obtained, resulting in the formulation of new
hypotheses. Examples for each of these aspects will be
discussed to demonstrate how they have improved our
understanding of fundamental processes leading to
deterioration of platelet functionality during
storage.
David Schriemer,
University of Calgary
Structural Biology with a Mass
Spectrometer
The proteome is a dynamic and responsive entity at
virtually every level of its definition, which suggests
that its analysis will never truly be complete.
Increasingly, our attention must shift to rigorous
characterization of its perturbation on a level beyond
composition, as a means to gain greater insights on complex
cellular functions. This characterization includes high
resolution analysis of structure and dynamics. While such
activities are not typically associated with the tools of
proteomics, there is a strong role for the mass
spectrometer in this area. In a fashion conceptually
related to NMR spectroscopy, restraints can be derived to
aid in structure/dynamic depictions of proteins, especially
their complexes. In my lab, we use various labeling
strategies to effect a link between protein structure and
mass, and develop hybrid MS/computational strategies for
characterizing complex structure and dynamics at
ever-increasing resolution.
In this talk, I will discuss our progress in developing a
proteomics-grade approach to the collection and mining of
hydrogen/deuterium exchange (H/DX)-MS data for these
purposes, and its application to data-directed docking
using various computational strategies. I will show by
example how such an enhanced approach can solve
longstanding structural problems in the area of microtubule
dynamics and drug discovery. I will also discuss some of
the inherent weaknesses to the methodology and present
preliminary data on new strategies designed to overcome
these weaknesses.
David Wishart,
University of Alberta
Using Proteomics to
Enable Metabolomics -- and Vice Versa
Proteomics
and metabolomics share more than the same “omics” suffix.
These two fields are intimately connected through the
shared technologies they both require to characterize and
annotate their chosen analytes. In this presentation
I will briefly review the technological and computational
needs in metabolomics and demonstrate how recent
developments in metabolomics have borrowed heavily from
concepts originally developed in proteomics. In
particular, I will describe the development of: 1)
novel isotope tagging techniques to characterize and
quantify metabolites via LC, MS and NMR; 2) the development
of “Mascot-like” search software and databases to identify
known metabolites; and 3) the creation of reference
compound libraries and reference standards to aid in
accurate quantification and quality control. I will
also show how developments in metabolomics can also aid a
number of facets in functional and structural proteomics,
including the deciphering of enzyme function and the
crystallization of novel proteins. The key message
for this presentation is that proteomics and metabolomics
can often be done in the same laboratory and that by
combining the information and ideas derived from both
fields one can gain a much greater understanding of
fundamental biological processes.