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Combinatorial
Chemistry & High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 8, Number 8, December 2005
Contents
Editorial Pp.657
Review Articles
Genome Projects and the Functional-Genomic Era
Pp. 659-667
Sascha Sauer, Zoltán Konthur and Hans Lehrach
[Abstract]
Gel-Based Versus Gel-Free Proteomics: A Review Pp.
669-677
Geert Baggerman, Evy Vierstraete, Arnold De Loof and Liliane
Schoofs
[Abstract]
Proteomics in Nutrition and Health Pp.
679-696
Martin Kussmann, Michael Affolter and Laurent B. Fay
[Abstract]
The Peptidomics Concept Pp. 697-704
Peter Schulz-Knappe, Michael Schrader and Hans-Dieter
Zucht
[Abstract]
Bioinformatics Challenges in Proteomics Pp.
705-715
Claudia C. Englbrecht and Axel Facius
[Abstract]
Datamining Methodology for LC-MALDI-MS Based Peptide
Profiling Pp. 717-723
Hans-Dieter Zucht, Jens Lamerz, Valery Khamenia, Carsten
Schiller, Annette Appel, Harald Tammen, Reto Crameri and Hartmut
Selle
[Abstract]
Research Papers
Prerequisites for Peptidomic Analysis of Blood Samples:
I. Evaluation of Blood Specimen Qualities and Determination
of Technical Performance Characteristics Pp. 725-733
Harald Tammen, Imke Schulte, Rüdiger Hess, Christoph
Menzel, Markus Kellmann and Peter Schulz-Knappe
[Abstract]
Prerequisites for Peptidomic Analysis of Blood Samples:
II. Analysis of Human Plasma after Oral Glucose Challenge
– A Proof of Concept Pp. 735-741
Harald Tammen, Rüdiger Hess, Imke Schulte, Markus
Kellmann, Annette Appel, Petra Budde, Hans-Dieter Zucht and
Peter Schulz-Knappe
[Abstract]
High-Throughput Biomarker Discovery and Identification
by Mass Spectrometry Pp. 743-755
Christoph Menzel, Vincent Guillou, Markus Kellmann, Valery
Khamenya, Michael Juergens and Peter Schulz-Knappe
[Abstract]
Towards Characterization of the Human Urinary Peptidome
Pp. 757-765
Michael Jürgens, Annette Appel, Gabriele Heine, Susanne
Neitz, Christoph Menzel, Harald Tammen and Hans-Dieter Zucht
[Abstract]
The Concept of Functional Peptidomics for the Discovery
of Bioactive Peptides in Cell Culture Models Pp.
767-773
Marco M. Jost, Petra Budde, Harald Tammen, Rüdiger
Hess, Markus Kellmann, Imke Schulte and Horst Rose
[Abstract]
Peptidomics Biomarker Discovery in Mouse Models of
Obesity and Type 2 Diabetes Pp. 775-781
Petra Budde, Imke Schulte, Annette Appel, Susanne
Neitz, Markus Kellmann, Harald Tammen, Rüdiger Hess and
Horst Rose
[Abstract]
Identification of Peptide Tumor Markers in a Tumor
Graft Model in Immunodeficient Mice Pp. 783-788
Harald Tammen, Karl Schorn, Hartmut Selle, Rudiger Hess,
Susanne Neitz, Rudolf Reiter and Peter Schulz-Knappe
[Abstract]
Peptide Sequence Prediction Supported by Correlation-Associated
Networks in Human Cerebrospinal Fluid Pp. 789-799
Jens Lamerz, Reto Crameri, Leonardo Scapozza, Thomas Mohring,
Hartmut Selle and Hans-Dieter Zucht
[Abstract]
Identification of Novel Biomarker Candidates by Differential
Peptidomics Analysis of Cerebrospinal Fluid in Alzheimer’s
Disease Pp. 801-806
Hartmut Selle, Jens Lamerz, Katharina Buerger, Andreas
Dessauer, Klaus Hager, Harald Hampel, Johann Karl, Markus
Kellmann, Lars Lannfelt, Jukka Louhija, Matthias Riepe, Wolfgang
Rollinger, Hayrettin Tumani, Michael Schrader and Hans-Dieter
Zucht
[Abstract]
The Future of Post-Genomic Biology at the Proteomic
Level: An Outlook Pp. 807-810
Reto Crameri, Peter Schulz-Knappe and Hans-Dieter Zucht
[Abstract]
Meet the Guest Editor Pp.811
[Abstract]
Abstracts
[Back to top]
Editorial
Life sciences in the last century have been affected by the
revolution in understanding the chemical nature of how genetic
information is stored, replicated, transcribed and translated.
The discovery of the DNA double helix by Watson and Crick
opened a new era for the rational understanding of fundamental
biological processes and initiating a cascade of progress
ranging from the discovery of the restriction enzymes, transformation
systems to transfer DNA into heterologous hosts, and development
of sophisticated sequencing techniques culminating in the
elucidation of the whole human genome. Although we are far
from understanding the organisation of a genome, the availability
of whole genome sequences facilitates the prediction of open
reading frames, protein sequences, the understanding of regulatory
and evolutionary processes, and the direct exploitation of
sequence knowledge for industrial applications. After reaching
the fundamental milestone of sequencing whole genomes, the
scientific community is now faced with the perhaps even bigger
challenge of understanding how genetic information is translated
into function. One of the basic lessons learned from the genome
projects is that humans and other organisms have far fewer
genes deposited in their DNA than had been assumed. In spite
of the tremendous impact of genomics on molecular biology
and medicine, the information encoded by genomes does not
represent more than a rough ground plan for an organism. This
information might restrict but does not describe the dynamics
of life processes. Gene activation, transcription and maturation
of mRNAs, translation, as well as post-translational modifications
give rise to a multitude of mature proteins and peptides with
structures which are not directly deducible from genome sequences.
The consequential development of new (global) research strategies,
as pointed out by Lehrach and co-workers in an essential review
in this issue, will characterize the post-genomic era. Classical
2D gel-based and gel-free proteomics (reviewed by Baggerman
et al.) represent a first step towards the simultaneous investigation
of all proteins expressed by a biological system under specific
physiological conditions. Proteomics, although suffering from
many limitations as discussed in detail in the review by Jussman
et al., has the potential to deliver comprehensive analyses
of the protein complement in biological fluids, cells, tissues,
and organisms. Proteomics evolved from technology platforms
aimed at identifying and characterizing single proteins to
a global approach addressing protein expression, protein-protein
interactions, structure and function. Peptidomics, reviewed
by Schulz-Knappe and co-workers, represents a restricted window
of proteomics dealing with small proteins and polypeptides
and aims to establish highly sensitive and reproducible technologies
for the identification of peptides of biological relevance.
Interestingly the concept of peptidomics, although an obvious
development of proteomics, evolved late as a global strategy
for peptide profiling and was first described in Combinatorial
Chemistry & High Throughput Screening (Vol. 4 (2), 2001,
207-217). Since both proteomics and peptidomics generate large
and complex amounts of data they depend on sophisticated bioinformatics
tools for data management and interpretation (see Englbrecht
and Facius in this issue). There are several features that,
when combined, affect the performance of the various proteomics
and peptidomics separation techniques and, therefore, the
accuracy of the analysis of data is of outmost importance.
The way from mass spectrometry based profiling of proteins
and peptides to their identification through data mining technologies
(summarized by Zucht et al.) is long but has great potential
for the discovery new biologically active peptides that might
be used in clinical laboratories as diagnostic tools. A prerequisite
for exploiting this potential is the demonstration of sensitivity
and reproducibility of the methods used in “proof of
concept” experiments, followed by experimental target
validation. A series of research communications presented
in this issue aims at demonstrating the performance of peptidomics
technology for the discovery of biologically relevant peptides.
These works show that peptidomics is a suitable technology
to investigate body fluids, cell cultures, and animal models.
Peptidomics allows fast and reliable profiling of complex
samples through robust methodological tools. The bioinformatics-based
analyses of complex data sets does not depend on any experimental
hypothesis but can help to generate hypotheses about relations
between a structural entity detected under a defined experimental
condition and other structural entities differentially subject
to parallel changes under the same conditions. However, the
path from the detection of targets with potential diagnostic
or therapeutic applications to target validation and to the
market will not be short.
Although a special issue covering such a vast field as proteomics
and peptidomics can never be complete, I hope that the contributions
enclosed in this issue will serve to catalyze research activities
in this fascinating and fast moving field. I am grateful to
all the authors for the time they invested in the realization
of this monograph, and I am confidently looking forward to
exciting progress in understanding the global processes governing
life.
Reto Crameri
Swiss Institute of Allergy and
Asthma Research (SIAF)
Obere Strasse 22
CH-7270 Davos
Switzerland
E-mail: crameri@siaf.unizh.ch
[Back to top]
Genome Projects and the Functional-Genomic Era
Sascha Sauer, Zoltán Konthur and Hans Lehrach
The problems we face today in public health as a result
of the - fortunately - increasing age of people and the requirements
of developing countries create an urgent need for new and
innovative approaches in medicine and in agronomics. Genomic
and functional genomic approaches have a great potential to
at least partially solve these problems in the future. Important
progress has been made by procedures to decode genomic information
of humans, but also of other key organisms. The basic comprehension
of genomic information (and its transfer) should now give
us the possibility to pursue the next important step in life
science eventually leading to a basic understanding of biological
information flow; the elucidation of the function of all genes
and correlative products encoded in the genome, as well as
the discovery of their interactions in a molecular context
and the response to environmental factors. As a result of
the sequencing projects, we are now able to ask important
questions about sequence variation and can start to comprehensively
study the function of expressed genes on different levels
such as RNA, protein or the cell in a systematic context including
underlying networks. In this article we review and comment
on current trends in large-scale systematic biological research.
A particular emphasis is put on technology developments that
can provide means to accomplish the tasks of future lines
of functional genomics.
[Back to top]
Gel-Based Versus Gel-Free Proteomics: A Review
Geert Baggerman, Evy Vierstraete, Arnold De Loof and Liliane
Schoofs
With the sequencing of the genome of over 150 organisms,
the field of biology has been revolutionised. Instead of studying
one gene or protein at the time, it is now possible to study
the effect of physiological or pathological changes on the
expressioin of all genes or proteins in the organism. Proteomics
aims at the simultaneous analysis of all proteins expressed
by a cell, tissue or organism in a specific physiological
condition. Because proteins are the effector molecules in
all organsims, it is evident that changes in the physiological
condition of an organism will be reflected by changes in protein
expression and/or processing. Since the formulation of the
concept of proteomics in the mid 90’s proteomics has
relied heavily on 2 dimensional gel electroforesis (2DGE)
for the separation and visualization of proteins. 2DGE, however,
has a number of inherent drawbacks. 2DGE is costly, fairly
insensitive to low copy proteins and cannot be used for the
entire proteome. Therefore, over the years, several gel-free
proteomics techniques have been developed to either fill the
gaps left by 2DGE or to entirely abolish the gel based techniques.
This review summarizes the most important gel-free and gel-based
proteomics techniques and compares their advantages and drawbacks.
[Back to top]
Proteomics in Nutrition and Health
Martin Kussmann, Michael Affolter and Laurent B. Fay
Proteomics, the comprehensive analysis of a protein complement
in a cell, tissue or biological fluid at a given time, has
been enabled by quantum leaps in mass spectrometric technology,
which allowed identification of large, involatile biomolecules.
Over the last two decades, this discipline evolved from the
sole delivery of protein identities to a platform, which reveals
clues to function through e.g. characterisation of protein
modifications and interactions as well as through quantitative
proteomics, i.e. the global comparison of protein amounts
between two defined biological states.
Proteomics is an integral part and key player in the family
of –omic disciplines as there are genomics (gene analysis),
transcriptomics (gene expression analysis) and metabolomics
(metabolite profiling). Considering the complexity, dynamics
and protein concentration range of any given proteome, proteomics
is the most challenging –omic discipline and requires
the most sophisticated analysis pipeline.
Proteomics represents an established technology in the pharmaceutical
industry mainly for biomarker and drug target discovery. The
potential of proteomics for research in the food industry
is increasingly being recognised and the employment of proteomic
approaches to nutrition and health issues is now emerging.
This review summarizes (i) major technological achievements
in mass spectrometry and proteomics, (ii) deliverables of
proteomics in the context of nutrition and health, and (iii)
applications of proteomics, and - if appropriate - transcriptomics
to the research fields of digestive health, obesity and diabetes,
immunity and allergy, probiotics, milk, and food preference.
[Back to top]
The Peptidomics Concept
Peter Schulz-Knappe, Michael Schrader and Hans-Dieter
Zucht
Peptides are a paramount example of how nature diversifies
from one single gene to release multiple, regulated functionalities
at the desired sites and time. To achieve this, peptides are
sequentially generated by a complex network of more than 500
proteases, acting at intracellular sites, upon secretion,
in extracellular environments, and, finally, serving (regulated)
degradation. This cycle of maturation, activation, and degradation
points out that the peptidome is mechanistically linked to
the proteome: the distribution between both is regulated by
proteases and counter-regulated by protease inhibitors. Given
the high diversity of peptides in living systems and their
involvement in key regulatory processes, a need for improved
peptide discovery, ideally combining peptide sequence identification
with peptide profiling, has emerged. Standard proteomic approaches
are not suitable for a systematic peptide analysis, since
they do not cover the low molecular mass window. The new direction
in proteomic research to analyse this “terra incognita”
is peptidomics. This novel concept aims at the comprehensive
visualization and analysis of small polypeptides, thus covering
the mass range between proteomics and metabonomics. The pacemakers
for the development of peptidomics technologies are modern
mass spectrometry and bioinformatics. They are ideally suited
for sensitive and comprehensive peptide analysis, especially
in combination with the massive information content of todays
genomic and transcriptomic databases. Given the high diversity
of native peptides in living systems, clinical chemistry and
modern medicine are the prime application areas. The discovery
of relevant peptide biomarkers and drug targets will strongly
benefit from peptidomics.
[Back to top]
Bioinformatics Challenges in Proteomics
Claudia C. Englbrecht and Axel Facius
A little after the genomic revolution had been celebrated,
it seemed as if a competition began to found new –omics
disciplines that ultimately all have the same goal, the understanding
of biological function. There are many similar definitions
for proteomics that can be summarized as follows: proteomics
is a large-scale study of structure and function of proteins
in an organism or cell. Importantly, the proteome is much
more variable than the genome through its interactions with
the genome and secondary modifications. It differs depending
on the tissue and stage in life-cycle. Hence, proteomics is
a very diverse discipline that uses a variety of experimental
set-ups and targets in order to elucidate function. Its dissociation
from other disciplines can only remain artificial. The bioinformatics
applied to proteomics are equally varied. In this review we
will focus mainly on a few areas of bioinformatics that seem
to us as particularly noteworthy or characteristic for proteomics
research, for example in 2DE analysis or mass spectrometry.
Another important task of bioinformatics is the prediction
of functional properties. We will summarize the approaches
taken in order to predict protein networks, which are based
on the extensive integration of several kinds of –omics
data. We will give a short overview of a demanding field in
computational biology, the analysis and prediction of protein
3D structures. In order to provide a broader perspective we
will close this review with a generalized description of activities
and databases in the realm of proteomics.
[Back to top]
Datamining Methodology for LC-MALDI-MS Based Peptide
Profiling
Hans-Dieter Zucht, Jens Lamerz, Valery Khamenia, Carsten
Schiller, Annette Appel, Harald Tammen, Reto Crameri and Hartmut
Selle
This report will provide a brief overview of the application
of data mining in proteomic peptide profiling used for medical
biomarker research. Mass spectrometry based profiling of peptides
and proteins is frequently used to distinguish disease from
non-disease groups and to monitor and predict drug effects.
It has the promising potential to enter clinical laboratories
as a general purpose diagnostic tool. Data mining methodologies
support biomedical science to manage the vast data sets obtained
from these instrumentations. Here we will review the typical
workflow of peptide profiling, together with typical data
mining methodology. Mass spectrometric experiments in peptidomics
raise numerous questions in the fields of signal processing,
statistics, experimental design and discriminant analysis.
[Back to top]
Prerequisites for Peptidomic Analysis of Blood Samples:
I. Evaluation of Blood Specimen Qualities and Determination
of Technical Performance Characteristics
Harald Tammen, Imke Schulte, Rüdiger Hess, Christoph
Menzel, Markus Kellmann and Peter Schulz-Knappe
Proteomics studies aiming at a detailed analysis of proteins,
and peptidomics, aiming at the analysis of the low molecular
weight proteome (peptidome) offer a promising approach to
discover novel biomarkers valuable for different crucial steps
in detection, prevention and treatment of disease. Much emphasis
has been given to the analysis of blood, since this source
would by far offer the largest number of meaningful biomarker
applications. Blood is a complex liquid tissue that comprises
cells and extra-cellular fluid. The choice of suitable specimen
collection is crucial to minimize artificial occurring processes
during specimen collection and preparation (e.g. cell lysis,
proteolysis). After specimen collection, sample preparation
for peptidomics is carried out by physical methods (filtration,
gel-chromatography, precipitation) which allow for separation
based on molecular size, with and without immunodepletion
of major abundant proteins.
Differential Peptide Display (DPD) is an offline-coupled
combination of Reversed-Phase-HPLC and MALDI mass spectrometry
in combination with in-house developed data display and analysis
tools. Identifications of peptides are carried out by additional
mass spectrometric methods (e.g. online LC-ESI-MS/MS). In
the work presented here, insights into semi-quantitative mass
spectrometric profiling of plasma peptides by DPD are given.
This includes proper specimen selection (plasma vs. serum),
sample preparation, especially peptide extraction, the determination
of sensitivity (i.e. by establishing detection limits of exogenously
spiked peptides), the reproducibility for individual as well
as for all peptides (Coefficient of Variation calculations)
and quantification (correlation between signal intensity and
concentration). Finally, the implications for clinical peptidomics
are discussed.
[Back to top]
Prerequisites for Peptidomic Analysis of Blood Samples:
II. Analysis of Human Plasma after Oral Glucose Challenge
– A Proof of Concept
Harald Tammen, Rüdiger Hess, Imke Schulte, Markus
Kellmann, Annette Appel, Petra Budde, Hans-Dieter Zucht and
Peter Schulz-Knappe
Mass spectrometric plasma analysis for biomarker discovery
has become an exploratory focus in proteomic research: the
challenges of analyzing plasma samples by mass spectrometry
have become apparent not only since the human proteome organization
(HUPO) has put much emphasis on the human plasma proteome.
This work demonstrates fundamental proteomic research to reveal
sensitivity and quantification capabilities of our Peptidomics
technologies by detecting distinct changes in plasma peptide
composition in samples after challenging healthy volunteers
with orally administered glucose.
Differential Peptide Display (DPD) is a technique for peptidomics
studies to compare peptides from distinct biological samples.
Mass spectrometry (MS) is used as a qualitative and quantitative
analysis tool without previous trypsin digestion or labeling
of the samples. Circulating peptides (< 15 kDa) were extracted
from 1.3 mL plasma samples and the extracts separated by liquid
chromatography into 96 fractions. Each fraction was subjected
to MALDI MS, and mass spectra of all fractions were combined
resulting in a 2D-display of > 2,000 peptides from each
sample.
Endogenous peptides that responded to oral glucose challenge
were detected by DPD of pre-and post-challenge plasma samples
from 16 healthy volunteers and subsequently identified by
nESI-qTOF MS. Two of the 15 MS peaks that were significantly
modulated by glucose challenge were subsequently identified
as insulin and C-peptide. These results were validated by
using immunoassays for insulin and C-peptide. This paper serves
as a proof of principle for proteomic biomarker discovery
down to the pM concentration range by using small amounts
of human plasma.
[Back to top]
High-Throughput Biomarker Discovery and Identification
by Mass Spectrometry
Christoph Menzel, Vincent Guillou, Markus Kellmann, Valery
Khamenya, Michael Juergens and Peter Schulz-Knappe
Native peptides and proteins are of increasing interest
in biomedical research because they hold promise to represent
a large number of useful diagnostic and therapeutic biomarkers.
Discovery attempts from patient samples have to deal with
the complexity of biology from a disease perspective as well
as with a high individual variability. High throughput screening
of samples is therefore the strategy of choice to detect relevant
peptidic biomarkers, and requires a high order of automation
particularly in the detection process. In this contribution,
a novel technical approach employing a fully automated MALDI-TOF/TOF
mass spectrometer is described. This approach combines high
throughput biomarker discovery with the identification of
corresponding endogenous peptides in one instrument and from
the same set of samples. The degree of automation allows the
analysis of thousands of chromatographic fractions corresponding
to up to one hundred patient samples per day. The applied
relative quantification via Differential Peptide Display¨
is performed in a label-free way and shows a dynamic range
of up to four orders of magnitude in the accessible peptide
concentrations. The typical limit of detection is in the mid-
to low-picomolar range for body fluids such as blood plasma,
urine and cerebrospinal fluid. Sequence assignment via MALDI–TOF/TOF
mass spectrometry is carried out either in an overview approach,
characterizing rapidly the peptide composition e.g. of a novel
sample, or in a directed approach, analyzing a list of biomarker
candidates deduced from statistically significant abundance
differences from the biomarker discovery process.
[Back to top]
Towards Characterization of the Human Urinary Peptidome
Michael Jürgens, Annette Appel, Gabriele Heine, Susanne
Neitz, Christoph Menzel, Harald Tammen and Hans-Dieter Zucht
Biomarker discovery in human urine has become an evolving
and potentially valuable topic in relation to renal function
and diseases of the urinary tract. In order to deliver on
the promises and to facilitate the development of validated
biomarkers or biomarker panels, protein and peptide profiling
techniques need high sample throughput, speed of analysis,
and reproducibility of results. Here, we outline the performance
characteristics of the liquid chromatography/MALDI-TOF-MS
based differential peptide display (DPD1) approach for separating,
detecting, abundance profiling and identification of native
peptides derived from human urine. The typical complexity
of peptides in human urine (resolution of the technique with
respect to detectable number of peptides), the reproducibility
(coefficient of variation for abundance profiles of all peptides
detected in biological samples) and dynamic range of the technique
as well as the lower limit of detection were characterized.
A substantial number of peptides present in normal human urine
were identified and compared to findings in four published
proteome studies. In an explorative approach, pathological
urines from patients suffering from post-renal-filtration
diseases were qualitatively compared to normal urine. In conclusion,
the peptidomics technology as shown here has a great potential
for high throughput and high resolution urine peptide profiling
analyses. It is a promising tool to study not only renal physiology
and pathophysiology and to determine new biomarkers of renal
diseases; it also has the potential to study remotely localized
or systemic aberrations within human biology.
[Back to top]
The Concept of Functional Peptidomics for the Discovery
of Bioactive Peptides in Cell Culture Models
Marco M. Jost, Petra Budde, Harald Tammen, Rüdiger
Hess, Markus Kellmann, Imke Schulte and Horst Rose
Detection and purification of novel bioactive peptides from
biological sources is a scientific task that led to a substantial
number of important discoveries. One major laborious approach
used is the repetitive stepwise separation of the test sample
into several fractions followed by the determination of their
bioactivity, until purity allows for sequence identification.
We tested whether functional peptidomics, a combination of
biological read-outs with differential peptide display (DPD)
is a suitable strategy to isolate bioactive peptides at lower
workload and with improved success. Additionally, we evaluated
the use of DPD to monitor the processing status of proinsulin
by inhibition of the insulin processing pathway. The rat insulinoma
cell line INS-1 stimulated either with 2 mmol/l or 10 mmol/l
glucose was used as model to generate differential peptide
displays. In parallel, the bioactivity of the supernatants
from the INS-1 cells was measured by glucose uptake and lipolysis
assays using the adipocyte cell line 3T3-L1. We were able
to quickly and elegantly trace the known activity of insulin
to increase glucose uptake and inhibit lipolysis. Following
re-chromatography of selected fractions, relevant peptides
were identified by DPD and bioassays: the rat insulin-1 precursor
and two different insulin peptides. We demonstrated in a semi-quantitative
fashion that inhibition of proinsulin processing leads to
accumulation of the insulin precursor, and reduced secretion
of insulin-1. Thus, we conclude that DPD is an attractive
support technology in peptide purification strategies aiming
to identify bioactive compounds, and is superior to ELISA
in discriminating between the processing status of insulin
and its precursor.
[Back to top]
Peptidomics Biomarker Discovery in Mouse Models of
Obesity and Type 2 Diabetes
Petra Budde, Imke Schulte, Annette Appel, Susanne Neitz,
Markus Kellmann, Harald Tammen, Rüdiger Hess and Horst
Rose
Type 2 diabetes mellitus (T2DM) is caused by the failure
of the pancreatic beta-cell to secrete sufficient insulin
to compensate a decreased response of peripheral tissues to
insulin action. The pathological events causing beta-cell
dysfunctions are only poorly understood and early markers
that would predict islet function are missing. In contrast
to immunoassays, unbiased proteomic technologies provide the
opportunity to screen for novel marker protein and peptides
of T2DM. An important subset of the proteome, peptides and
peptide hormones secreted by the pancreas are deregulated
in T2DM. The mass range of peptides and small proteins (1-20
kDa) is only sufficiently targeted by peptidomics, a combination
of liquid chromatographic and mass spectrometric (MS) peptide
analysis. Here, we describe the application of isotope label-free
quantitative peptidomics to display and quantify relevant
changes in the level of pancreatic peptides and peptide hormones
in a preclinical model of T2DM, the Lepob/Lepob mouse. The
amino acid sequence of statistical relevant top candidates
was determined by MS/MS fragmentation or Edman degradation.
The comparison of lean versus obese mice revealed increased
levels of islet-specific peptides that can be divided into
the following categories 1) the major islet peptide hormones
insulin, amylin and glucagon; 2) proinsulin and C-peptide
and 3) novel processing products of secretogranin, glucagon
and amylin. Furthermore, we found increased levels of proteins
and peptides implicated in zymogen granule maturation (syncollin)
and nutritional digestion. In summary, our findings demonstrate
that peptidomics is a valid approach to screen for novel peptide
biomarkers.
[Back to top]
Identification of Peptide Tumor Markers in a Tumor
Graft Model in Immunodeficient Mice
Harald Tammen, Karl Schorn, Hartmut Selle, Rudiger Hess,
Susanne Neitz, Rudolf Reiter and Peter Schulz-Knappe
The medical demand for useful biomarkers is large and still
increasing. This is especially true for cancer, because for
this disease adequate diagnostic markers with high specificity
and sensitivity are still lacking. Despite advances in imaging
technologies for early detection of cancer, peptidomic multiplex
techniques evolved in recent years will provide new opportunities
for detection of low molecular weight (LMW) proteome biomarker
(peptides) by mass spectrometry. Improvements in peptidomics
research were made based on separation of peptides and/or
proteins by their physico-chemical properties in combination
with mass spectrometric detection, respectively identification,
and sophisticated bioinformatic tools for data analysis.
To evaluate the potential of serological tumor marker detection
by differential peptide display (DPD) we analyzed plasma samples
from a tumor graft model. After subcutaneous injection of
HCT-116 cells in immunodeficient mice and their growth to
a palpable tumor, plasma samples were analyzed by DPD. The
comparison of obtained mass spectrometric data allows discovery
of tumor specific peptides which fit well into the biological
context of cancer pathogenesis and show a strong correlation
to tumor growth.
The identified peptides indicate events associated with hyper-proliferation
and dedifferentiation of cells from an epithelial origin,
which are typical characteristics of human carcinomas. We
conclude that these findings are a “proof of principle”
to detect differentially expressed, tumor-related peptides
in plasma of tumor-bearing mice
[Back to top]
Peptide Sequence Prediction Supported by Correlation-Associated
Networks in Human Cerebrospinal Fluid
Jens Lamerz, Reto Crameri, Leonardo Scapozza, Thomas Mohring,
Hartmut Selle and Hans-Dieter Zucht
During the course of biosynthesis, processing and degradation
of a peptide, many structurally related intermediate peptide
products are generated. Human body fluids and tissues contain
several thousand peptides that can be profiled by reversed-phase
chromatography and subsequent MALDI-ToF-mass spectrometry.
Correlation-Associated Peptide Networks (CAN) efficiently
detect structural and biological relations of peptides, based
on statistical analysis of peptide concentrations. We combined
CAN with recognition of probable cleavage sites for peptidases
and proteases in cerebrospinal fluid, resulting in a model
able to predict the sequence of unknown peptides with high
accuracy. On the basis of this approach, identification of
peptide coordinates can be prioritized, and a rapid overview
of the peptide content of a novel sample source can be obtained.
[Back to top]
Identification of Novel Biomarker Candidates by Differential
Peptidomics Analysis of Cerebrospinal Fluid in Alzheimer’s
Disease
Hartmut Selle, Jens Lamerz, Katharina Buerger, Andreas
Dessauer, Klaus Hager, Harald Hampel, Johann Karl, Markus
Kellmann, Lars Lannfelt, Jukka Louhija, Matthias Riepe, Wolfgang
Rollinger, Hayrettin Tumani, Michael Schrader and Hans-Dieter
Zucht
The objective of this work was the application of peptidomics¨1
technologies for the detection and identification of reliable
and robust biomarkers for Alzheimer’s disease (AD) contributing
to facilitate and further improve the diagnosis of AD. Using
a new method for the comprehensive and comparative profiling
of peptides, the differential peptide display¨ (DPD),
312 cerebrospinal fluid (CSF) samples from AD patients, cognitively
unimpaired subjects and from patients suffering from other
primary dementia disorders were analysed as four independent
analytical sets. By combination with a cross validation procedure,
candidates were selected from a total of more than 6,000 different
peptide signals based on their discriminating power. Twelve
candidates were identified using mass-spectrometric techniques
as fragments of the possibly neuroprotective neuroendocrine
protein VGF and another one as the complement factor C3 descendent
C3f. The combination of peptide profiling and cross validation
resulted in the detection of novel potential biomarkers with
remarkable robustness and a close relation to AD pathophysiology.
[Back to top]
The Future of Post-Genomic Biology at the Proteomic
Level: An Outlook
Reto Crameri, Peter Schulz-Knappe and Hans-Dieter Zucht
Drug discovery and early-stage drugs and biomarkers development
is a continuous adaptation and maturation process. The cycle
of changes based on new findings is coupled with shifts in
research priorities and make this part of pharmaceutical research
a challenging endeavour. Over the last years, the emphasis
on genomics has shifted to proteomics, the science of understanding
how proteins translate gene information into function, and
metabonomics, the science of small metabolites that are further
apart from genomic projects. Proteomics describes the analysis
of the protein complement of a biological sample with respect
to temporal and spatial resolution. This technology is based
on separation of complex protein mixtures by 2D gel-electrophoresis,
in gel digest and mass spectrometric analysis of the protein
fragments. Proteomics has been recently flanked by peptidomics,
a new research direction aimed at the comprehensive analysis
of small (1-20 kDa) polypeptides, thus covering the gap between
proteomics and metabonomics. The refinement of peptidomics
is based on an essential paradigm related to modularity and
diversity. Peptides are a paramount example of how one single
gene can release multiple functionalities. We can expect fast
progress in understanding protein and peptide networks from
a systems biology approach ending in the discovery of new
peptide targets. However, the way from a complex sample to
potential diagnostic and therapeutic targets will depend on
technological developments and from the ability to discriminate
true disease-related signals from false positive and negative
signals, and the way from target discovery to target validation
will not be short.
[Back to top]
MEET THE GUEST EDITOR
Reto Crameri
Head Molecular Allergology
Professor of Molecular Immunology
Swiss Institute of Allergy and Asthma Research (SIAF)
Obere Strasse 22
CH-7270 Davos
Switzerland
Reto Crameri studied microbiology and biochemistry at the
Swiss Federal Institute of Technology (ETH) Zürich, where
he completed his PhD on genetics of industrial microorganisms
in 1980. He was a research scientist and head of the antibiotic
research group until the end of 1982 at the same institution.
Thereafter, he joined Biogen SA in Geneva as senior scientist
in molecular biology until end of 1986. After a short period
as head of the Swiss Radon Project at the Paul Scherrer Institute
(1987-1989), he moved at the Swiss Institute of Allergy and
Asthma Research in Davos as head of the Division of Molecular
Allergology. In addition he became guest Professor for Molecular
Immunology at the University of Salzburg, Austria, in 1996.
Dr. Crameri is on the Editorial Board of Allergy, Biochemical
Journal, and International Archives of Allergy and
Immunology and is or has been a reviewer of grant applications
for the Österreichiser Nationalfonds (FWF), the Swiss
National Science Foundation (SNF), The Wellcome Trust, and
The Eli and Edythe L. Broad Foundation, among others. Besides
contributions to the fields of allergology and immunology,
he recently played an important role as a founder of ImVisioN
GmbH & Co. KG, Hannover, Germany, a spin-off company dealing
with recombinant vaccines.
The research interests of Dr. Crameri include development
of phage display and robot-based high throughput screening
technology for fast identification of medically important
target molecules, studies on the mechanisms of innate immunity,
development of diagnostic tools based on molecular biology,
elucidation of molecular structures, and mass spectrometric-based
profiling of peptides and small proteins from human body fluids
and tissues. Some recent publications are cited below.
SELECTED PUBLICATIONS
Kodzius R.; Rhyner C.; Konthur Z.; Buczek D.; Lehrach H.;
Walter G.; Crameri R. Rapid identification of allergen-encoding
cDNA clones by phage display and high-density arrays. Combin.
Chem. High Throughput Screen. 2003,
6, 147-154.
Fossa A.; Alsoe L.; Crameri R.; Funderud S.; Gaudernack G.;
Smeland E. B. Serological cloning of cancer/testis antigens
expressed in prostate cancer using cDNA phage surface display.
Cancer Immunol. Immunother. 2004,
53, 431-438.
Andersson A.; Rasool O.; Schmidt M.; Kodzius R.; Flückiger
S.; Zagari A.; Crameri R.; Scheynius A. Cloning, expression
and characterization of two new IgE-binding proteins from
the yeast Malassezia sympodialis with sequence similarities
to heat shock proteins and manganese superoxide dismutase.
Eur. J. Biochem. 2004, 271,
1885-1894.
Akdis M., Verhagen J., Taylor A., Karmaloo F., Karagiannidis
C.; Crameri R.; Thunberg S.; Deniz G.; Valenta R.; Fiebig
H.; Kegel C.; Disch R.; Schmidt-Weber C.B.; Blaser K.; Akdis
C. A. Immune responses in healthy and allergic individuals
are characterized by a fine balance between allergen-specific
T regulatory 1 and T helper 2 cells. J. Exp. Med.
2004, 199, 1567-1575.
Kussebi F.; Karamloo F.; Rhyner C., Schmid-Grendelmeier P,:
Salagianni M.; Manhart C.; Akdis M.; Soldatova L.; Markovic-Housley
Z.; Von Beust B.R.; Kündig T.; Kemeny D.M.; Blaser K.;
Crameri R.; Akdis C. A. A major allergen gene-fision protein
for potential usage in allergen-specific immunotherapy. J.
Allergy Clin. Immunol. 2005, 115,
323-329.
Schmid-Grendelmeier P.; Flückiger S.; Disch R.; Trautmann
A., Wüthrich B.; Blaser K.; Scheynius A.; Crameri R.
IgE-meidated and T cell-mediated autoimmunity against manganese
superoxide dismutase in atopic dermatitis. J. Allergy
Clin. Immunol. 2005, 115, 1068-1075.
Lamerz J.; Selle H.; Scapozza L.; Crameri R.; Schulz-Knappe
P.; Mohring T.; Kellmann M.; Khameina V.; Zucht H.D. Correlation-associated
peptide networks of the human cerebrospinal fluid. Proteomics
2005, 5, 2789-2798.
Crameri R. The potential of proteomics and peptidomics for
allergy and asthma research. Allergy 2005,
60, 1227-1237.
Limacher A.; Kloer D. P.; Flückiger S.; Folkers G.;
Crameri R.; Scapozza L. The crystal structure of Aspergillus
fumigatus cyclophilin reveals 3D domain swapping of a
central element. Structure 2005 (in
press).
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