Characterizing the Cogs in
the Machinery of Life
Now that the human genome sequence is complete, the quest to extract beneficial
knowledge from it is on. One of the most promising, active areas of exploration
lies in the human proteome--the global expression of proteins, those marvelous
strings of amino acids responsible for all human biologic processes. Proteins
are life, and the recently developed ability to study them on a large scale, quantitatively
and qualitatively, is known as proteomics. The human proteome may never be completely
solved in the same way the genome was. The genome is relatively static, and presented
a finite end point. The proteome is dynamic, changing constantly with time and
conditions, with proteins interacting to form networks and pathways to respond
to stimuli and carry on the endless business of cellular function. The challenge
of completely mapping the proteome is widely considered to be several orders of
magnitude greater than that of the genome. The picture is so complex and so dynamic
that some proteomics experts question the very concept of the existence of a measurable
human proteome. Famed genomicist J. Craig Venter put this doubt succinctly when
he told the 5 April 2001
Wall Street Journal that "there ain't no such
thing as a proteome."
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The
proteome is constantly changing. Technologically, you’re always
trying to hit a moving target.
--
B. Alex Merrick
NIEHS
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Nevertheless, there can be no debate that proteomics is poised to deliver vast
amounts of useful information about physiologic function at the subcellular,
cellular, organic, and systemic levels, yielding profound new insights into
disease and drug mechanisms, the effects of environmental exposures, and much
more. Although a comprehensive map of the entire human proteome may never be
accomplished, protein maps of human organs, glands, and fluids and of entire
less-complex organisms are within sight, and major efforts are under way to
document many of those proteomes.
Evolution of Proteomics
In large measure, proteomics has emerged in parallel fashion with the other
"-omics" fields such as transcriptomics and metabonomics. The technologies,
methodologies, and grand ambitions of the Human Genome Project have rapidly
proliferated and now permeate virtually every area of the life sciences.
Just as the advent of genomics brought the ability to discover large numbers
of genes quickly, proteomics was born when technologic advances allowed scientists
to widen their focus from the painstaking isolation and identification of single
proteins to a more comprehensive view of the entire protein complement expressed
in a given cell line, tissue, or organism. However, proteomics researchers employ
their own unique mix of tools, approaches, and skills to address the questions
they seek to answer.
Although the term "proteomics" did not exist until 1994, when Australian postdoctoral
student Marc Wilkins coined it, the practice of the science has been going on
since the mid-1970s. Two milestone technologic breakthroughs facilitated the
ability to look at multiplicities of proteins, both of which, although much
refined, are still in wide use today in laboratories around the world.
At its core, proteomics is all about separation and identification--the process
of taking a sample of interest, separating out all of the proteins therein,
and then identifying them. The first major breakthrough, which was a great leap
forward in separation, took place in 1975 with the introduction of two-dimensional
gel electrophoresis (2DE).
With this method--still the first step in many proteomics experiments--proteins
from a sample are separated on a polyacrylamide gel according to their mass
and charge, which, along with intensity, are what provide the spectrum that
makes up a protein's distinctive signature. The more abundant the protein, the
larger and more intensely staining the spot on the gel.
The only problem is that 2DE, while it allows separation and visualization
of the protein complement, does little or nothing to address identification.
Regardless, the advent of 2DE was so exciting that in 1980 it spawned the proposal
of a Human Protein Index project--an effort to catalog all human proteins and
then use that knowledge to define the genome (although Congress considered the
project, it was never funded, and advances in genomics soon bypassed the idea).
The second major breakthrough, which really brought proteomics into its own,
was the arrival of two crucial techniques in the 1980s that made possible the
use of mass spectrometry (MS) to identify proteins: matrix-assisted laser desorption/ionization
(MALDI) and electrospray ionization (ESI). These methods allow protein samples
to be ionized for analysis in a mass spectrometer, producing a pattern called
a mass spectrum. These mass spectra--which often number in the thousands for
a given sample--can then be used to positively identify proteins or protein
digests (strings of peptides or protein fragments produced when the proteins
are ionized) through the automatic querying of protein databases. Unidentified
or novel proteins can be analyzed through further MS runs or by other techniques.
"ESI and MALDI were a quantum leap," says William Pierce, a professor of pharmacology,
toxicology, and chemistry at the University of Louisville School of Medicine.
The subsequent development of time-of-flight (TOF) detection, which expanded
the range of ionic molecular weights detectable by MS instruments, brought further
analytic capabilities to MALDI. Today, MALDI-TOF is in widespread use in proteomics
laboratories.
A dazzling panoply of MS refinements and enhancements, as well as the development
of other technologies, now facilitate the application of proteomic techniques
to a highly diversified universe of research pursuits. Virtually every proteomics
laboratory, whether it's connected with government, academia, or industry, seems
to have its own favored technologic approach, and many have developed their
own in-house methods, along with customized bioinformatics software, to help
sort through and make sense of the massive amounts of data their systems generate.
"I think we need more than one platform to be able to adequately do service
to measuring proteins in a global fashion," says B. Alex Merrick, head of the
Proteomics Group at the NIEHS National Center for Toxicogenomics (NCT). "The
proteome is constantly changing. Technologically, you're always trying to hit
a moving target." Merrick cautions, however, that "because proteins have so
many properties, or attributes, and we have the potential to measure them, it
spawns many, many platforms, and technologically we haven't sorted out which
platforms are the best ones."
The general feeling among proteomics researchers is that the field is on the
verge of consolidating years of method development into a flood of knowledge.
"I think we're about to transition out of the age of explorers in proteomics
to the age of applications," says Daniel Liebler, a professor of biochemistry
and director of proteomics at the Vanderbilt University School of Medicine.
"Proteomics techniques are not going to be done just as demonstrations of powerful
technology, but will really be integrated into studies in basic laboratory science,
animal and nonanimal models of diseases and environmental exposures, and actually
in human clinical studies as well."
Proteomics in Action: Cancer Detection
Thanks to progress in clinical proteomics, someday soon a simple blood test
could hold the key to early diagnosis of certain cancers. That is one of the
many goals of the Clinical Proteomics Program, a joint research effort that
is codirected by biochemist Emanuel Petricoin of the U.S. Food and Drug Administration
(FDA) and pathologist Lance Liotta of the National Cancer Institute (NCI).
The group has developed a method of identifying protein patterns in blood
serum that is potentially indicative of the presence of a wide range of diseases.
Their initial study, published 16 February 2002 in The Lancet, focused
on ovarian cancer, which presently has both a poor late-stage survival rate
and a poor early detection rate--a deadly combination fostering an urgent need
for better diagnostic tests, especially for women at high risk for developing
the disease.
The investigators used surface-enhanced laser desorption/ionization TOF (SELDI-TOF),
a variation on MALDI-TOF that incorporates protein microarrays and is particularly
well suited to detecting patterns of proteins in samples. First, a "training"
set of known, unblinded samples was run through the instrument--in this case,
serum samples from both healthy women and women with ovarian cancer. With the
high throughput of the equipment, spectra for each sample--each one containing
15,200 data points, or individual pieces of information--were quickly generated.
Next, the raw data were processed by a unique bioinformatics system that incorporates
a form of artificial intelligence called a genetic algorithm. The genetic algorithm
compares the patterns of protein expression in the diseased samples to those
in the healthy samples, looking for those patterns that optimally discriminate
between the two. The algorithm learns as it goes in a process that involves
hundreds of millions of pattern combinations and comparisons. The end product
is a pattern of unidentified proteins--in this case, five--that precisely distinguishes
healthy samples from diseased ones.
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Proteomics
techniques are not going to be done just as demonstrations of powerful
technology, but will really be integrated into studies in basic laboratory
science, animal and nonanimal models of diseases and environmental exposures,
and actually in human clinical studies as well.
--
Daniel Liebler
Vanderbilt University School of Medicine
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The next step was to run a set of known but blinded samples through the same
process, and then compare the results to assess the predictive power of the
patterns. In this study, the investigators achieved a sensitivity of 100%--that
is, all of the cancerous samples were correctly identified, with no false negatives--and
a specificity of 95%, meaning only 5% of the identifications were false positive.
This was vastly superior to the 35% positive predictive value in the same samples
of cancer antigen 125, the present gold standard clinical biomarker.
Subsequent technical refinements (which included a switch to a much higher-resolution
and more stable mass spectrometer, and incorporation of advanced spectral quality
control methods) have improved the system's sensitivity and specificity to 100%
in a larger blinded set of ovarian cancer and high-risk samples. The team is
currently enrolling participants in a clinical trial to test their methodology
in detecting recurrence of ovarian cancer.
Although the proteins in the discriminatory pattern generated by this method
are at least initially unidentified, Petricoin says that is beside the point.
"We as scientists want to understand what the nature of the beast is, and we're
hunting that down," he says. "We're already making great progress to that end.
But we don't see identity as necessary for its use in diagnostics."
Two of the world's largest reference laboratory companies apparently agree.
Quest Diagnostics and LabCorp have sublicensed the technology from Correlogic
Systems (which developed the initial genetics algorithm and licensed the technology
from the U.S. government), and plan to start offering the proteomic test as
an ovarian cancer screening tool for women at high risk by the end of 2003.
Initially, they will market the procedure under the FDA's "home brew" provision,
which allows the companies to perform the service only in their own validated
laboratories.
Petricoin is optimistic that proteomic pattern diagnostics could impact medical
diagnostics in a big way. "This is a different type of diagnostic paradigm,"
he says. "[It] completely changes and turns on its head the normal tried-and-true
route--which we would suggest is failing--of looking at discovery biomarkers.
. . . I think if either our or LabCorp/Quest's efforts are successful, it's
really going to throw a gauntlet down on a completely different type of diagnostic
procedure being used in the clinic. That could have reverberations throughout
disease detection, period."
The FDA/NCI group is applying this proteomic technique in similar studies
of breast, lung, pancreatic, esophageal, brain, and prostate cancers, as well
as efforts to detect cancer drug cardiovascular toxicity before symptoms occur,
and to assess the effectiveness of molecularly targeted cancer drugs.
Another Approach: Cancer Profiling
The Mass Spectrometry Research Center at the Vanderbilt University School
of Medicine, headed by Richard Caprioli, the Stanley Cohen Professor of Biochemistry,
also is pursuing methods of distinguishing diseased tissue from healthy tissue,
particularly in cancer. But Caprioli's group takes a very different approach,
in which identification of the proteins in the affected tissues themselves,
rather than in plasma, is central. It's called tissue proteome profiling, and
it appears to be a powerful new tool for both diagnosis and prognosis.
Tissue proteome profiling has several advantages, including the ability to
accurately detect factors such as life expectancy and tumor aggressiveness.
Tissue proteome profiling could also directly identify potential targets for
drug intervention, as well as contribute to understanding of mechanisms of the
disease.
In their most complete study to date, published 9 August 2003 in The Lancet,
Caprioli and his coworkers concentrated on lung tumors. They took hundreds of
lung tumor biopsy samples and analyzed their protein complements via MALDI MS,
looking at several spots on each sample, each of which generated thousands of
signals in a specific pattern of proteins. Then, using a series of bioinformatics
tools, they correlated the tissue proteome profiling information with known
information about the individual patients, some of whom had already died of
their disease.
"We found that at the first level, we could find unique sweeps of proteins
that helped us actually classify the disease," says Caprioli. "So if you take
the biggest set of lung tumors, nonsmall cell lung carcinomas, we could
further classify them as adenocarcinomas, squamous cell carcinomas, and so on."
Of course, pathologists can do the same, but Caprioli says they didn't stop
there. "We asked, 'Can we correlate these patterns with the life expectancy
or the prognostic value of these diseases?' And it turned out to be of very
high accuracy." He says the researchers could tell from the protein pattern
which patients would go on to survive for long periods of time, and which patients
would die of cancer--"so the aggressiveness of the disease was apparent in the
protein profile."
They were further able to pick out with approximately 80% accuracy those patients
whose tumors had metastasized, causing nodal involvement and the often inoperable
development of secondary tumors. There is presently no other method of making
such a crucial clinical prediction.
Although appropriately cautious to point out that these results were in just
one type of tumor study, Caprioli is excited about the possibility of using
protein patterns to identify types of tumors that have an aggressive posture
for nodal involvement. "It begins to get you out of just diagnosing to actual
patient care, so that the clinician can now identify a high-risk group and make
the appropriate therapeutic decisions," he says.
Caprioli's group is nearing completion of a similar study of brain tumors
with similar results in terms of the power of tissue protein profiling for prognostication.
They're also looking at diabetes mellitus, cardiac and pulmonary diseases, and
several other conditions. Caprioli asserts that this platform, along with other
clinical proteomics work, ultimately constitutes an entry point into the field
of individualized medicine, centered around the concept that each patient's
disease is unique at the molecular level. "It's a whole new way of looking at
things," he says. "There's no doubt in my mind that as we collectively learn
more and more about the molecular ways of diagnosing disease, of predicting
disease progression, that this individualized way of looking at diseases will
become more and more common."
Toxicoproteomics: Mechanisms and Biomarkers
Liebler's main focus is toxicoproteomics. His group concentrates on understanding
how reactive intermediates produce deleterious effects by modifying proteins.
These unstable chemical species enter a cell as a result of environmental exposures
and tend to bind to proteins or DNA, modifying their properties in an injurious
way and forming new biomolecules known as adducts.
Using a form of MS called tandem MS, or MS/MS, along with a novel algorithm
and proprietary bioinformatics software called Scoring Algorithm for Spectral
Analysis, Liebler and his group are able to analyze the mass spectra of peptides
to establish their sequences, the positions of any modifications, and, by mapping
that information back onto the entire protein sequence, the sites of modification
in the protein itself. Ultimately, two major questions are addressed: What are
the protein targets of reactive intermediates? And what are the cellular responses
to protein modification?
Answers to these questions will shed light on some of the most important avenues
of contemporary research in toxicology. Liebler sees the biggest near-term payoff
of this type of work as coming in two general areas. One area is the understanding
of mechanisms of toxicity. The other is the identification of biomarkers of
exposure. "If we can figure out what the targets of some of these environmental
compounds are or what reactive intermediates come from environmental stimuli
or stresses, by understanding mechanisms we then know what components of the
cell or tissues might be amenable to some kind of protective intervention,"
he says.
As proof of principle, Liebler's team published a study in the June 2002 issue
of Chemical Research in Toxicology documenting their system's ability
to map hemoglobin adducts of the aliphatic epoxides, a group of common industrial
chemicals. The team is most interested in investigating biomarkers of oxidative
stress, the damaging phenomenon implicated in many disease processes and often
the result of environmental exposure. "What we would like to do is identify
some of the most abundant of these reactive intermediates that are formed under
representative conditions either in vitro or in animal models in vivo,
where we can manipulate oxidative stress," says Liebler.
Pierce's biomolecular MS lab at Louisville is involved in similar functional
proteomics work. His group looks at subsets, or small clusters, of functionally
interactive proteins. They isolate post-translational modifications, any of
more than 100 different types of changes that can be made to proteins by a variety
of factors after their original creation. (This partially accounts for the vastly
larger number of proteins than genes.) Pierce's group also works to develop
or validate biomarkers in cases of specific environmental or xenobiotic exposures
and those agents' interactions with nucleic acids and proteins.
In a collaboration with Louisville professor of medicine Aruni Bhatnagar,
Pierce and colleagues are looking at a very large, ubiquitous set of chemicals,
the aldehydes, which form adducts with proteins, potentially contributing to
cardiovascular disease. Aldehydes are not just environmental contaminants, but
are also naturally present in food and are intermediates in human metabolism.
By identifying aldehyde-induced adducts and elucidating how they might influence
protein function, the team hopes to characterize a novel mechanism involved
in hypertension, stroke, and other forms of cardiovascular disease.
Pierce is also working on a project with university associate professor of
medicine James Summersgill, investigating the interactions of the microorganism
Chlamydia pneumoniae with the cardiovascular system. Just as Helicobacter
pylori has been implicated in gastric ulcers, there is a theory that microorganisms
in the cardiovascular system could cause systemic infection, leading to plaque
development and atherosclerosis. "We study the chlamydial proteome and look
at changes in it and how th at might be reflected in the production of products
that then stimulate atherosclerotic lesions," says Pierce.
Like all proteomics practitioners, he is enthusiastic about the possibilities
that lie ahead in the field. "The infinite variety of states of proteins in
the cell will give us the opportunity, more so than in genomics, to uncover
new mechanisms in biology," he says. "In certain aspects you're looking at a
dynamic system that is growing and changing, and we can actually 'catch biology
happening.' And because of that, we'll find new mechanisms and be more likely
to develop new ways to look at mechanisms or affect them."
NIEHS researchers are also delving into the field of proteomics. Merrick and
the NCT Proteomics Group, working in partnership with Kenneth B. Tomer, who
heads the NCT Mass Spectrometry Group, aid the center's efforts to discover
more and better information about the adverse effects of chemicals and toxic
compounds. Merrick's group works mainly in expression proteomics experiments
with animals. "We want to be able to evaluate the effects of chemicals in experimental
animals under the most controlled conditions possible," he says, "so that we
can separate out the true effects of the chemicals from the nonspecific 'noise'
effects that you always see with these types of technologies."
Among other projects, Merrick's group uses SELDI to examine the mechanisms
of action of two key proteins involved in cell growth and cell death--p53 and
NF
B.
"p53 is often regarded as one of the 'master switches' of life and death and
cell growth within the cell," says Merrick. "In the same sense, NFB
is a 'master switch' for inflammation and immune response. In these two proteins,
we're looking for specific markers, specific states that would distinguish them
in terms of their being activated or deactivated in association with a particular
disease state or state of cellular function."
In the 3 April 2001 issue of Biochemistry, the Merrick and Tomer groups
reported the results of their MS research on p53. They were able to isolate
the entire protein from the cell for comprehensive MS analysis for the first
time, in an effort to shed light on how the fine structure of the protein influences
its ability to control cellular life and death. It has long been suspected that
phosphorylation, a type of post-translational modification, may be involved
in the process. The group discovered six specific phosphorylation sites on the
protein, one of which, Ser(315), was particularly phosphorylated. Unraveling
the mystery of how p53 exerts its "master switch" control over cellular mortality
would be an important advance in biology, and this study constitutes a major
step toward that discovery.
The group is also undertaking a number of clinical projects in neurodegenerative
disease and cancer, taking advantage of access to blood and serum samples from
NIEHS epidemiologic activities. Serum proteomic analysis has much to tell, according
to Merrick. "The soluble proteins within serum or plasma can be reflective of
a disease state, or of toxicity or injury to a particular disease site, whether
it be in heart disease or liver disease," he explains. "So proteomics can shine
in analysis of serum or plasma because of the nature of disease, in that you
may either have release of a biomarker from a particular organ, or there may
be indications of a repair process going on with serum or plasma that you can
[detect in the bodily fluids]."
Toxicoproteomics can also be used to discover previously unknown sites within
the cell, says Merrick. "When you're dealing with proteins, you're dealing with
time and space," he says. "These proteins occupy a certain amount of space within
tissues or cells, and to be able to isolate these subcellular portions that
are important targets of either therapy or of toxicity is an area where proteomics
can make a special contribution."
Calcium, Oxidation, and Aging
At the Pacific Northwest National Laboratory in Richland, Washington, researcher
Thomas Squier practices proteomics as part of the laboratory's systems biology
approach, which integrates information from all of the -omics disciplines to
first determine how a cell functions and then develop predictive models. The
lab's Biomolecular Systems Initiative, which includes its proteomics work, is
part of the U.S. Department of Energy's Genomes to Life program, which aims
at uncovering biologic solutions for major environmental issues such as clean
energy production, removal of excess carbon dioxide from the atmosphere, and
remediation of contaminated environments.
Squier's group concentrates on analyzing calcium regulation in cells and how
oxidative stress can trigger adaptive mechanisms, resulting in post-translational
modifications to key calcium sensor proteins. Calcium maintains a 10,000-fold
gradient in cellular systems and is the key player in the signaling that modulates
energy metabolism. Changing calcium levels are responsible for much of the cell's
sensing of the environment. By identifying post-translational modifications
of the key calcium sensor proteins (changes such as methionine oxidation and
protein nitration), potentially important new biomarkers of exposure can be
isolated. For example, the lab has identified the calcium signaling protein
calmodulin as a major target of oxidative stress, as described in the January
2003 issue of Chemical Research in Toxicology. This discovery could contribute
significantly to understanding of adaptive cellular responses to environmental
exposures, particularly in how repair and maintenance systems are triggered.
Aging is an important factor in cellular adaptive ability as well. Aging is
a major risk factor for most diseases and for sensitivity to environmental exposures,
says Squier. "In aging," he says, "the key regulatory proteins get oxidized,
and their oxidation slows metabolism down. We speculate that this is an adaptive
mechanism to maintain this balance between reactive species and cell function."
Ultimately, this work on the detection of post-translational modifications
of key sensor proteins could lead to the development of microarray-based assays
that would rapidly analyze a person's antioxidant status. In terms of applications,
Squier says, being able to quickly identify changes in protein expression and
discern what post-translational modifications happen is going to provide a very
high level of information about the health of an individual, which in turn could
lead to greatly enhanced medical treatment.
Proteomics Initiatives and Databases
Considering the enormous challenges and opportunities posed by proteomics,
it's unsurprising that there are several collaborative proteomics initiatives
under way at the national and international levels. Perhaps the best known of
these campaigns is the Human Proteome Organisation (HUPO), an international
body intended to encourage large-scale analysis of the human proteome. HUPO
seeks to consolidate national and regional proteome organizations into a worldwide
research consortium. "Proteomics cannot be fully grasped and developed without
a major international organized effort, which HUPO intends to facilitate," says
Samir Hanash, the organization's president and a professor of pediatrics at
the University of Michigan.
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In
certain aspects you’re looking at a dynamic system that is growing
and changing, and we can actually ‘catch biology happening.’
And because of that, we’ll find new mechanisms and be more likely
to develop new ways to look at mechanisms or affect them.
-- William Pierce
University of Louisville School of Medicine
|
|
HUPO has established a goal of mapping 5,000 human proteins, and is coordinating
and standardizing research in a variety of pertinent areas. Its major projects
include analysis of specific regions of the body--the Human Plasma Proteome
Project, the Human Liver Proteome Project, and the Human Brain Proteome Project.
Another major project is the Proteomics Standards Initiative, which aims to
define community standards for presentation of proteomics data. Still other
projects include initiatives involving new proteomics technologies, cell models
and tissue, bioinformatics, and the development of a collection of standardized,
high-quality antibodies for every human protein. HUPO's shared resources, data,
and establishment of standardized protocols and reporting guidelines should
contribute substantially to the understanding of disease processes and chemical
exposures.
The Human Proteomics Initiative seeks to comprehensively annotate all known
human proteins, which means parsing out each protein's function, domain structure,
subcellular location, post-translational modifications, variants, similarities
to other proteins, and protein sequence polymorphisms. This ambitious project
is sponsored by the Swiss Institute of Bioinformatics and the European Bioinformatics
Institute, the keepers of one of the most widely used protein sequence databases,
Swiss-Prot.
Virtually all proteomics experiments involve accessing and querying protein
databases as an integral step in the process, allowing the identification and
characterization of detected proteins and peptides. That vital link between
data and knowledge should be greatly enhanced by the establishment of the United
Protein Database, or UniProt. Funded in October 2002 by a three-year, $15 million
grant subsidized primarily by the National Human Genome Research Institute along
with five other institutes and centers of the NIH, UniProt will combine the
resources of Swiss-Prot and two other major annotated protein databases, the
European Bioinformatics Institute's TrEMBL and the Protein Information Resource's
Protein Sequence Database. By January 2005, UniProt will be fully operational
and available to all users free of charge.
In the world of proteomics, the main action often centers around interactions,
molecular complexes, and pathways. The Biomolecular Interaction Network Database
serves as a comprehensive, publicly accessible repository for data and software
tools related to those critical biomolecular functions. The database is administered
by blueprint WORLDWIDE, a nonprofit organization cofounded for that purpose
by IBM and MDS Proteomics of Toronto, Canada.
Another important resource in the protein database arena has recently been
launched as a joint project between researchers at The Johns Hopkins University
in Baltimore, Maryland, and the Institute of Bioinformatics in Bangalore, India.
By the end of 2003, the Human Protein Reference Database is expected to contain
comprehensive entries on 10,000 human proteins, including domain architecture,
post-translational modifications, interaction networks, and disease associations.
The information in this database has been manually extracted from the literature
by biologists who read, interpret, and analyze the published data.
To spur the progress of clinical proteomics, in 2002 the National Heart, Lung,
and Blood Institute launched a major initiative that created 10 special centers
of proteomics research at academic institutions across the country. The seven-year,
$157 million program is designed to accelerate the development of innovative
technologies to characterize healthy and diseased heart, lung, blood, and sleep
processes. Says the institute's proteomic program administrator Susan Old, "This
should speed the delivery of potential new clinical applications from research
into practice." The centers will investigate protein profiling, interactions,
and post-translational modifications as they relate to a variety of conditions,
including cardiovascular disease, autoimmune disease, airway inflammation, and
cystic fibrosis.
The development of better tools and better knowledge of structural proteomics
is the goal of the Protein Structure Initiative, a 10-year project funded by
the National Institute of General Medical Sciences and launched in 2000 with
an open-ended budget. Currently in its pilot phase, the initiative aims to determine
the three-dimensional structure of 10,000 unique proteins, while dramatically
reducing the time and costs involved in the process. By 2005, each of nine centers
is expected to be able solve the structure of 100200 proteins annually.
By grouping proteins into structural families, "the initiative will develop
a catalog of all the protein structures that exist in nature," said Marvin Cassman,
then director of the National Institute of General Medical Sciences, at the
time the initiative was launched. "We expect that it will yield major biological
findings that will improve our understanding of health and disease."
Proteomics data are also expected to play a large role in the Chemical Effects
in Biological Systems (CEBS) knowledge base being developed by the NCT. CEBS
is designed to exhaustively document the toxic effects of chemicals in the environment
and will be fully searchable by compound, structure, toxicity, pathology, gene,
gene group, single-nucleotide polymorphism, pathway, and network. The knowledge
base will be accessible by the public, and will be a major contributor to progress
in the fields of toxicoproteomics and toxicogenomics.
Pieces
of the Proteomics Puzzle |
Proteomics
encompasses several different subdisciplines, each with its own unique
approach and its own contribution to the overall quest to glean knowledge
from the proteome. |
Expression
Proteomics
In expression (or profiling) proteomics, researchers seek to discover and
quantify significant differences in the totality of expressed proteins between
known samples--often diseased versus nondiseased or exposed versus unexposed.
These differences appear as patterns that can have a very high degree of
predictive power, whether the proteins in the pattern are identified (as
some experts contend is necessary) or remain unidentified (which others
argue is sufficient). Expression proteomics studies yield hypotheses that
are then confirmed or refuted by other methods. Clinical proteomics investigations,
which seek to apply proteomics knowledge directly to medical practice, typically
employ expression proteomics methods. |
Functional
Proteomics
Functional proteomics encompasses a wide variety of studies involving subsets
of proteins. These studies seek to analyze and characterize specific functions,
including signaling pathways, interactions, disease mechanisms, and biomarkers
of disease or environmental exposures. In this field, hypotheses are tested
rather than developed, and protein identification is vital to success. |
Structural
Proteomics
Structural proteomics concentrates on mapping the structure of protein complexes
or those proteins present in a specific cellular organelle. Such information
can provide valuable insights into cellular architecture, which greatly
influences cellular function. X-ray crystallography and structural modeling
by computational biology are the main methods utilized to unravel these
extremely complicated systems. |
Toxicoproteomics
In toxicoproteomics, the full range of proteomics methods and technologies
are used in efforts to uncover the cellular and subcellular mechanisms at
work in response to xenobiotic exposures. Researchers in this area are particularly
interested in discovering biomarkers of exposure. |
Proteomics Prognostications
Scratch your average proteomics investigator and you will reveal an optimist
just under the sober scientific surface. The excitement is palpable; the visions
are grand. But all in the field agree that for proteomics to fulfill its lofty
promise, certain key developments must take place, several of which are well
on their way to fruition.
Technologic progress must continue and accelerate. Many in the field are anxious
to see the replacement of the notoriously laborious 2DE method of protein separation
with a more automated, high-throughput approach, such as antibody microarrays
or isotope-coded affinity tags. All wish for further improvements and refinements
in MS equipment and bioinformatics, as well as development of other technologies
that could contribute to progress in the field.
"I think MS is becoming increasingly powerful, and we haven't yet realized
the full power of these tools," says Liebler. "On the other hand, I think MS-based
proteome analyses will give way to other kinds of less high-tech approaches,
using perhaps some variants of array technologies: arrays of antibodies or aptamers
[basic nucleic acid equivalents of antibodies] and perhaps small molecules that
recognize proteins--little lab-on-a-chip devices that would be suitable for
analysis of some components of proteins." There are a lot of competing technologies,
he says, and it's hard to say what's going to work--"but if the last ten years
have taught us anything, it's that we should be prepared to be surprised, regularly."
Philosophically, practitioners are confident that the knowledge gleaned from
proteomics will ultimately converge and integrate with advances in the other
-omics fields to evolve into a more holistic systems biology discipline with
the ability to understand the processes and mechanisms of life in a truly global
fashion. "We tend to think of ourselves as proteomics people, or genomics people,
or lipids people, and in fact the cell and tissue only exist because all of
these things are integrated," says Caprioli. "How these things all relate to
one another is what's going to give us the key [to a more comprehensive understanding
of systems biology]."
Researchers believe that proteomics will begin to make a tangible difference
in medicine and environmental health quite soon. Merrick, for example, believes
that within five years, there will be perhaps two or three key public databases
that will offer access to gene and protein expression experiments that are done
in a standardized way, and researchers will be able to query those databases
for use in predicting human health responses to various environmental interactions.
In 10 years, he says, "I believe that proteomics will be able to go right into
the clinic, in terms of diagnostics and evaluation of blood and serum in a way
that clinical chemistry can't approach or compete with. Typically, when you
get blood drawn, you get maybe twenty or thirty analyses. . . . I think in the
future this will just be dwarfed by the amount of useful information that will
be derived at the proteomic level."
Petricoin is even less guardedly optimistic. He predicts that in five years
"a patient will be able to have a pathophysiological portrait performed by high-throughput
protein-based technologies that can read out hundreds of thousands of end points
at once, and be able to provide the clinician a snapshot of what's going on
in that organism." Within a decade, he says, will come the development of high-throughput
proteomics coupled with artificial intelligencetype systems, with nanotechnology,
and even with nano-intelligence systems, allowing clinicians to harvest information
and deliver tailored therapeutics based on what's happening in the serum, plasma,
and tissue of any given patient who visits the doctor. "It's really going to
revolutionize the way in which molecular medicine is performed," says Petricoin.
"It's going to happen."
Ernie Hood
Proteomics
Resources |
| Groups
and Initiatives |
|
Human
Proteome Organisation (HUPO)
http://www.hupo.org/
An international
research consortium intended to encourage large-scale analysis of the
human proteome |
|
Human
Proteomics Initiative
http://www.expasy.org/sprot/hpi/
Joint effort of the
Swiss Institute of Bioinformatics and the European Bioinformatics Institute
that seeks to comprehensively annotate all known human proteins |
|
National
Heart, Lung, and Blood Institute Proteomics Initiative
A seven-year, $157
million program to accelerate the development of innovative technologies
to characterize healthy and diseased heart, lung,
blood, and sleep
processes in 10 special centers of proteomics research across the country
|
|
Protein
Structure Initiative
http://www.structuralgenomics.org/
A 10-year project
funded by the National Institute of General Medical Sciences to determine
the three-dimensional structures of 10,000 unique proteins, while dramatically
reducing the time and costs involved in the process |
| Databases |
|
Biomolecular
Interaction Network Database
http://www.blueprint.org/
A comprehensive,
publicly accessible repository administered by blueprint WORLDWIDE for
data and software tools related to critical biomolecular functions |
|
Chemical
Effects in Biological Systems Knowledge Base
http://www.niehs.nih.gov/nct/cebs.htm
National Center for
Toxicogenomics database that will exhaustively document the toxic effects
of chemicals in the environment and be fully searchable by compound, structure,
toxicity, pathology, gene, gene group, single-nucleotide polymorphism,
pathway, and network |
Human
Protein Reference Database
http://www.hprd.org/
Joint project of
The Johns Hopkins University and the Institute of Bioinformatics that
is expected to eventually contain comprehensive entries on 10,000 human
proteins, including domain architecture, post-translational modifications,
interaction networks, and disease associations |
|
Protein
Sequence Database
http://pir.georgetown.edu/pirwww/dbinfo/pirpsd.html
A comprehensive annotated
protein sequence database in the public domain, maintained by the Protein
Information Resource, that contained more than 283,000 entries as of November
2003 |
|
Swiss-Prot
http://us.expasy.org/sprot/
A curated protein
sequence database developed by the Swiss Institute of Bioinformatics and
the European Bioinformatics Institute that strives to provide a high level
of annotation, a minimal level of redundancy, and high level of integration
with other databases |
TrEMBL
http://www.ebi.ac.uk/trembl/
A database maintained
by the European Bioinformatics Institute that contains the translations
of all coding sequences present in the European Molecular Biology Laboratory's
Nucleotide Sequence Database that are not yet integrated into Swiss-Prot
|
|
United
Protein Database
http://www.uniprot.org/
With $15 million
in funding from six NIH institutes and centers, will combine the resources
of Swiss-Prot, TrEMBL, and the Protein Sequence Database. |
