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Review of the
5th Sienna Meeting – “From Genome to
Proteome: Functional Proteomics”
by Dr. Pratik Jagtap
The
5th Sienna Meeting – “From Genome to
Proteome: Functional Proteomics” --- held from the 2nd to 5th of
September was attended by over four hundred
participants. Ninety-one oral presentations were given and the poster session
gave an opportunity to overview 161 posters.
This meeting has a
tradition of setting trends in the field of proteomics. From the naming of
the field of ‘’Proteomics’’ by
Marc Wilkins (1994), to the introduction of novel sample
solubilization methods by Thierry Rabilloud (1996); from the
introduction of the concept of Molecular
scanner (1998) to the introduction of laser-capture microdissection and 2D
gel analysis software (2000) –-this meeting always breaks new grounds.
The opening lecture
was delivered by
Renato Dulbecco wherein he summarized the development of biological
science from genetics to genomics to proteomics and the new insights that
these developments provide in the understanding of biological systems.
The plenary session
on technical aspects covered the recent advances in the new methodologies
used in proteomics. Christoph Eckerskorn from Tecan Proteomics described
the advantages of freeflow electrophoresis as a prefractionation method.
He described the use of zonal freeflow electrophoresis in order to reveal
a closer view of the mitochondrial proteome. Tim Nadler from Applied
Biosystems, described the advances in the molecular scanner project with
particular reference to integration of multidimensional LC, SDS PAGE and
the scanner.
Angelika Gorg highlighted her work on perfecting 2D-gel
electrophoresis with special emphasis on narrow range IPG strips and
protein separation in the alkaline pH range. Maurizio Bruschi from
Giannina Gaslini Institute added a new variation to the theme of 2DE
protocol while using Soft IPGs and equilibrating strips before IEF.
Bengt
Bjellqvist from Amersham Biosciences, addressed the problem of streaking
and irreproducibility during IEF runs in the basic range. He described the
use of hydroxy ethyl disulfide as a reducing agent to separate proteins in
the alkaline pH range to provide a streak free 2D gel.
Ben Herbert addressed a similar problem of artifactual modifications
and the effect of electrophoresis, alkylation and alkaline pH on the
quality of protein spots.
Scott Patterson
chaired the session on protein characterization involving Nicole Packer
from Proteome Systems Limited, who described the advances in the field of
glycoproteomics. She discussed the development of
Glycosuite dB -- a database for identification of
oligosaccharides by using LC-ESI-MS or glycan mass fingerprinting of
enzymatically-cleaved oligosaccharides from 2D gel spots. Ronald Annan`s (Glaxo
Smithkline) talk focused on investigating biologically-relevant
phosphorylation using differential phosphopeptide mapping.
Thierry Rabilloud described the detection of overoxidation of cysteine
to cysteic acid during oxidation of peroxiredoxin as a potentially useful
marker for oxidative damage to cells.
The first oral
session on mass spectrometry and related technologies included a talk by
Martin Larsen ( University of Southern Denmark) who discussed the use of
hydrophobic graphite columns which results in retaining of post-translationally
modified peptides and a better sequence coverage and S/N ratio after MS
analysis. Kris Gevaert (Ghent University, Belgium) discussed the use of
COFRADIC (COmbined FRActionation by
DIagonal Chromatography) for small sample proteomic analysis to a variety
of samples including hepatocytes, E. coli, plasma and human
platelets. William Hancock from Thermofinnigan described the use of laser
capture microscopy and LC-MS to identify biomarkers. Takahashi Manabe from
Ehime University in Japan revealed the power of use of a stepwise approach
for analysis of protein complex formation. He used non-denaturing and
denaturing methods in 2DE to identify almost 60 IgG binding proteins.
Malcom Ward from King’s College in London, described his study on
hyperphosphorylation of Tau protein in Alzheimer’s disease.
In an oral
presentation session focusing on human disorders, Michael Harrington from
Huntington Medical Research Institute discussed the use of proteomics to
study migraine. In the study, CSF, blood and urine were subjected to
periodic proteomic analysis after triggering migraine in patients. Odile
Carette from Hospital University of Geneva described the power of the use
of SELDI - TOF MS for identifying novel Alzheimer’s disease markers. Robin
Wait from Kennedy Institute in London described the comparative proteomic
analysis of cartilage tissue from osteoarthritic and unaffected
individuals.
Hans-Jürgen
Thiesen from University of Rostock touched upon the need for assessing the
mRNA and protein levels in biological samples in order to validate
clinical relevance of ''omic'' data sets. Monique Slijper from Utrecht
University described the study of network of serine/threonine
phosphorylation induced by double stranded DNA breaks using B cells of
ataxia telangiectasia patients as a model system.
Dolores Cahill from Max-Planck Institute for Molecular Genetics described
her impressive work on generation
of a uniclone high-density protein array, which consists of 10,000
non-redundant foetal brain cDNA human proteins.
Plenary session with
the theme “protein quantitation” involved lectures on quantitative
proteomic analysis by non-2D gel based methods. Peter James from Lund
University described the use of separation of peptides (cleaved by
non-specific proteases) by single or multidimensional HPLC to study
membrane protein expression. Ruedi Aebersold
from Institute for Systems Biology stressed the need to develop newer
statistical models to study and validate the huge amount data generated by
''omic '' studies.
Dale Patterson from
Applied Biosystems delivered a seminar on the use of a novel acid
cleavable ICAT reagent to study the phenomenon of nonsense mediated mRNA
decay. The cells were treated with NMD2 protein (a key protein that
affects mRNA of almost 800 genes) and were subjected to comparative
proteomic analysis with untreated cells.
In the plenary
session of protein-protein interactions, Dario Neri from ETH in Zurich
discussed the details of generation of human antibody library and its
applications. Bernhard Kuster (Cellzome) described
their work on yeast using the TAP/ MS wherein
2660 proteins were networked. Along with the MUDPIT approach and MDS
method a total of 3498 different proteins have been networked in protein
complexes. Alma Burlingame from University of California described
his laboratory’s approach of deciphering the nuclear pore complex using
MALDI-TOF/ TOF MS.
“Pharmaceutical and
clinical applications” included a talk by Leigh Anderson from Plasma
Proteome Institute and he described the plasma proteome as the largest
proteome and one with widest range. Scott Patterson shared Celera’s vision
and their plans to identify small molecular therapeutic targets and the
identification of normal and diseased markers.
Keith Rose, the CSO of Geneprot, described their approach to identify
small proteins involved in coronary artery disease.
In oral
presentations on pharmacology and toxicology, Nicole Verills (Australian
Proteome Analysis Facility) discussed acquired mutations in gamma-actin
gene and its association with drug resistance in childhood acute
lymphoblastic leukemia. Jennifer Van Eyk from Queen’s University in Canada
revealed a large diversity of PTMs in the cardioprotective mechanisms of
myocardial preconditioning. Raiyin Chu from Aventis Pharma described the
identification of peroxisome proliferation markers by using SELDI. Per
Andren from Amersham Biosciences described the power of use of the nanoLC
ESI Q-TOF MS approach to identify endogenous neuropeptides in
neurodegenerative diseases such as the Parkinson’s disease.
In the oncology
session, Xue-Min Zhang from National Center of Biomedical Analysis
described the identification of four novel proteins that could be involved
in apoptosis. Cecilia Sarto from Derio Hospital in Milan suggested that
the degree of aquaporin glycosylation could be used in prognosis in renal
cell carcinoma.
The bioinformatics
oral session, chaired by Amos Bairoch, touched upon software development.
Matthias Berth from Decodon, described the use of position indexing for
spot matching with “Delta2D”. Patricia
Hernandez from Swiss Institute of Bioinformatics, described the
development of POPITAM – an ant colony optimisation algorithm-inspired and
driven software set for Tandem Mass Spectral analysis. Guido Bologna from
Swiss Institute of Bioinformatics described the use of neural network
ensembles to predict myristoylation at the N-terminal of peptides. Peter
Hornbeck from Cell Signalling Technology described the development of a
curated phosphosite database – a useful
tool for signal transduction studies.
Adriano Aguzzi, from the Institute of Neuropathology (Zurich) gave an
impressive account on their laboratory’s work on the aspect of
neuroinvasion in murine prion disease. He stressed on the immunological
aspect of the work – such as the involvement of B cells and the
involvement of the complement system.
The plenary session
on biological and biomedical applications, Pier Giorgio Righetti from
University of Verona, addressed the prion solubilisation problem and
demonstrated that there is a huge amount of microheterogeneity in the
PrPSc isoforms. Lukas Huber from University of Innsbruck, demonstrated the
power of organellar proteomics in identifying late endocytosome marker and
its interacting pathways. Julio Celis from Danish Center for Genome
Research described the use of identifying markers among low-grade
papillary TCCS and its correlation with recurring form of breast cancer.
Pranav Sinha from Universitats Klinikum Charite, used
differentially-expressed tumor proteins to transfect cell lines and then
assess the effect their role in chemoresistance. Michael Dunn from
Institute for Psychiatry in London highlighted the development of a
non-invasive analytical method for studying post-cardiac transplant
rejection using proteomics. Guido Grandi from Chiron Vaccines described
the use of a combined approach that uses bacterial cell FACS analysis
using sera against specific proteins and proteomic analysis to identify
surface vaccine candidates. Brad Walsh from Proteomeca, used a proteomic
approach to correlate quality markers of wheat such as heat resistance and
dough quality. Paul Haynes from Torrey Mesa Research
Institute
gave an account of their exhaustive work on identifying the rice proteome.
The study involved using both 2D-GE and MudPIT method to identify a total
of 2528 different proteins. This study, along with others, demonstrated
that both these methods are complementary.
Michel Desjardins delivered the main lecture regarding phagocytosis in
the genomic era. Using an organellar proteomic approach, his laboratory
identified 350 phagosomal proteins. He also showed the active role of ER
in phagosomal membrane biogenesis.
Christian Pasquali
from Serono, demonstrated the use of proteomic approaches to study the
specificity and sensitivity of phosphoinositide-protein interactions to
identify the downstream effectors of PI3K signaling in the oral session on
eukaryotic cells and tissues. Elisabetta Gianazza from University of Milan
discussed about the importance of redox reactions in regulating key
metabolic enzymes. Cecilia Gelfi studied muscle proteomes to demonstrate
the altered protein expression in Tibetans as a means to study the genetic
correlation of altitude adaptation. Albert Heck from Utrecht University
used metabolic labeling of C. elegans proteins by feeding them on
15N labelled E. coli. Protein was extracted from the labeled and
unlabelled organisms and 2D-gels were analyzed.
In the prokaryotes
session, Stuart Cordwell from Australian Proteome Analysis Facility,
Sydney demonstrated the power of using IEF-based prefractionation methods
to analyse proteins with extreme pI and Mw from Helicobacter pylori.
He also discussed the use of affinity-based fractionation methods to
analyze specific functional properties of proteins. Kathleen Champion from
Genentech, discussed the E.coli “immunome”– a proteome composed of
400 proteins that are highly reactive with goat antisera against E.
coli cell lysate. Erin Schiller from Pfizer described the use of E.
coli 2D-gel “signatures” (protein profile patterns) as a means to
analyze the effect of known antibiotics. The database resulting from the
profiling studies can be used to study the nature of novel antibiotics or
mechanism of a compound. Michael Gohar from Aventis CropSciences analyzed
the effect of a transcriptional regulator – PlcR – on the extracellular
proteome in Bacillus sps.
The Plenary session
on bioinformatics and knowledge bases consisted of a talk by Peter
Jungblut from Max-Planck Institute for Infection Biology, who presented
the collection of
proteomic databases for 2D-gel and ICAT analysis and a functional
classification database for Mycobacterium tuberculosis and
Helicobacter pylori. Zeev Smilansky highlighted the improvement in
Compugen’s Zeta 3 software to Z4000 system. John Cottrell applied the
Mann and Wilm “error tolerant” mode for peptide sequence tag method for
the careful
interpretation of MS/MS data. Amos Bairoch from Swiss institute
of
Bioinformatics
gave an impressive account of two annotation projects, viz.,
Human Proteomics Initiative (HPI) and the
High Quality Automated Microbial Annotation of Proteomes (HAMAP). HPI
annotates all known human sequences and provides information on
post-translational modifications, spliced isoforms, polymorphisms and 3D
structural data. He stressed the need to carry out cDNA sequencing
projects along with eukaryotic genome sequencing projects for easier
protein prediction. The HAMAP project aims to automatically annotate
proteins originating from bacterial and archaeal genome sequencing
projects.
In the study of
complexity plenary session entitled, Jan van Oostrum from Novartis Pharma
gave an overview on the proteomic methods that are used for validation and
identification of mechanisms.
John Yates III gave an account of the use of the MUDPIT method and
shotgun proteomics. He stressed that the use of multiple chromatography
and the use of specific and non-specific proteases could help in the
identification of PTMs especially in complex proteomes such as lens.
Rainer Hillenbrand from Novartis Pharma stressed the need to use
highly-specific antibodies in identifying surrogate markers of drug
effects.
Mathias Uhlen from
Royal Institute of Technology (Sweden) delivered the closing lecture on a
novel affinity ligand-based proteomic approach. The work was primarily
based on the use of
affibodies-–robust “artificial antibodies” generated using an
in vitro combinatorial approach--to detect the human chromosome 21
protein products. The affibodies were used to analyze specific protein
expression in tissues and localization of proteins.
Dennis Hochstrasser adjourned the meeting remarking that there
was a need to return back to biology and answer relevant questions by
applying advances in technology. The proceedings of the Meeting will be
covered in the January 2003 issue of
Proteomics.
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