Download Proteomic pearl diving versus systems biology in cell physiology

Editorial Focus
Am J Physiol Cell Physiol 306: C634–C635, 2014;
doi:10.1152/ajpcell.00010.2014.
Proteomic pearl diving versus systems biology in cell physiology. Focus
on “Proteomic mapping of proteins released during necrosis and apoptosis
from cultured neonatal cardiac myocytes”
Mark A. Knepper
Epithelial Systems Biology Laboratory, Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes
of Health, Bethesda, Maryland
Address for reprint requests and other correspondence: M. A. Knepper,
Epithelial Systems Biology Laboratory, Systems Biology Center, NHLBI,
NIH, Bethesda, MD 20892-1603 (e-mail: [email protected]).
C634
employ all of the information in large-scale data sets by
identifying patterns in the data, relating these patterns to
existing knowledge about cellular function. This objective
subsumes much of what is commonly termed “systems biology.” Both the pearl diving approach and the systems biology
approach are “discovery” modalities. They differ in that the
former discovers interesting proteins for further study, while
the latter discovers co-regulated sets of proteins that point to
particular cell biological processes that may be involved in the
physiological response.
The bulk of the paper by Marshall and colleagues examines
models of necrosis in primary cultures of rat neonatal cardiac
myocytes. They use state-of-the-art protein mass spectrometry
to identify proteins released into the medium in response to
H2O2-mediated induction of necrosis and apply both analytical
approaches described above, viz. proteomic pearl diving and
systems biology, to interpret their results. From their pearl
diving, the authors found a number of known markers of
necrosis such as lactate dehydrogenase (LDH) and the DNAbinding protein Hmgb1. In addition, a number of other interesting proteins were enriched in the media relative to controls,
such as ␣-actinin, HSP90, and Trim72. The last is a musclespecific protein that plays an important role in membrane
repair. The proteins found are considered potential biomarkers
of myocyte necrosis, information that could be of considerable
use in two ways, viz. possible clinical applications and use in
future studies to understand mechanisms of cardiac injury such
as seen in ischemia-reperfusion injury. Thus, proteomic pearl
diving proved successful. Antibodies recognizing several of
the marker proteins discovered by the authors were utilized to
confirm a limited set of observations from the proteomic
studies. Proteomics investigators have recently suggested that
antibody-based assays are inferior to targeted mass spectrometry for confirmation studies, in part because many antibodies
are far less specific than is protein mass spectrometry (1).
Marshall et al. implicitly provide a counterargument to this
suggestion, showing that when reliable antibodies are available, they can be used for cost-effective extensions of a study.
In the case of the current paper, the authors used immunoblotting to show that the same marker proteins appear in the
medium after induction of necrosis by a different agent, ␤-lapachone instead of H2O2, thus supporting the idea that many of
the proteins found are necrosis markers rather than redox
markers.
The authors also employed a systems biology approach,
identifying several pathways that contain significantly greater
numbers of necrosis-associated proteins than would be expected by a chance selection from a control data set. This type
of analysis has a major advantage in the physiological context
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IN THE EARLY DAYS of the 21st century, the completion of
genome sequencing projects for multiple species provided a
bounty of new information for physiological investigations.
The availability of comprehensive genome sequence data has
also made possible new large-scale approaches to the study of
biology that are particularly promising for cell physiology and,
therefore, of particular interest to readers of this journal. These
methods include DNA microarrays, deep sequencing techniques, and large-scale proteomics using protein mass spectrometry. Since proteins are responsible for most cellular
functions, proteomics seems particularly appropriate for studies of cell physiology. Recognizing this, the Editors of AJPCell Physiology initiated a Call for Papers that use mass
spectrometry methodology for studies of cell physiology. This
call resulted in a series of excellent publications that includes
metabolomics and DNA array studies in addition to proteomics (2, 3, 5, 7, 8). The latest in this series is the article
by Marshall et al. (4) in this issue. This report uses protein
mass spectrometry to identify proteins released from cultured neonatal cardiac myocytes in models of cell death.
While no one would debate the ability of proteomics (and
other large-scale methods) to generate large amounts of data
that are relevant to important physiological questions, there is
considerable skepticism among cell physiologists about the
utility of such data. This skepticism exists largely because of
the perceived absence of adequate methods for finding relevant
information in large data sets and relating it to existing knowledge. Many (if not most) cell physiologists are most comfortable exploring the function of their favorite cells one gene or
protein at a time, taking advantage of methods that manipulate
the abundance of a given protein in a cell or mutating one or
more amino acids. Based on this reductionist perspective, when
faced with a study presenting large data sets such as that of
Marshall et al. (4), investigators may be inclined to search the
expanse of reported data and try to identify a single protein or
a few proteins that warrant further study. Or, reviewers may
ask authors to “pick a particular protein from the data set and
study it further.” Such an approach, viewed as proteomic “pearl
diving,” is often effective when it identifies a key protein
previously not recognized to play a role in the physiological
process under consideration. However, doing so has a large
down-side, namely, by relegating most of the results to a
virtual data-dumpster, it wastes most of the information generated. Thus, the major challenge in physiological proteomics
is the development and utilization of methods that consider and
Editorial Focus
C635
ACKNOWLEDGMENTS
The author is supported by the intramural budget of the National Heart,
Lung and Blood Institute (NHLBI) (Project ZO1-HL001285, M. A. Knepper).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author.
AUTHOR CONTRIBUTIONS
M.A.K. drafted manuscript; edited and revised manuscript; approved final
version of manuscript.
REFERENCES
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AJP-Cell Physiol • doi:10.1152/ajpcell.00010.2014 • www.ajpcell.org
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in that it identifies cellular processes rather than individual
proteins whose connection to the physiology may be obscure.
In addition, the systems biology approach tends to be statistically more robust: False positives are less likely since the
process identified depends on quantification of multiple proteins versus the individual proteins identified with pearl diving.
An example from Marshall et al. is their top-ranked pathway
(Table 1), viz. “aryl hydrocarbon receptor signaling.” The
discovery of this pathway points to a possible role in cardiac
necrosis for a set of very interesting basic helix-loop helix
transcription factors that are already known to play roles in
blood pressure regulation and cardiac physiology (9) as well as
redox regulation (6). Although implication of aryl hydrocarbon
receptor signaling is hypothetical at this point, it is easy to
imagine experiments to test its role in the cellular decision
process between the life and death of cardiac myocytes.