Circulation Research. 2002;90:380-389
doi: 10.1161/hh0402.105757
(Circulation Research. 2002;90:380.)
© 2002 American Heart Association, Inc.
Applied Proteomics
Mitochondrial Proteins and Effect on Function
Mary F. Lopez,
Simon Melov
From Proteome Systems (M.F.L.), Woburn, Mass; and Buck Institute for Age Research (S.M.), Novato, Calif.
Correspondence to Mary F. Lopez, PhD, Proteome Systems, 14 Gill St, Woburn, MA 01801. E-mail mary.lopez{at}proteomesystems.com
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Abstract
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The identification of a majority of the polypeptides in mitochondria
would be invaluable because they play crucial and diverse roles
in many cellular processes and diseases. The endogenous production
of reactive oxygen species (ROS) is a major limiter of life
as illustrated by studies in which the transgenic overexpression
in invertebrates of catalytic antioxidant enzymes results in
increased lifespans. Mitochondria have received considerable
attention as a principal sourceand targetof ROS.
Mitochondrial oxidative stress has been implicated in heart
disease including myocardial preconditioning, ischemia/reperfusion,
and other pathologies. In addition, oxidative stress in the
mitochondria is associated with the pathogenesis of Alzheimers
disease, Parkinsons disease, prion diseases, and amyotrophic
lateral sclerosis (ALS) as well as aging itself. The rapidly
emerging field of proteomics can provide powerful strategies
for the characterization of mitochondrial proteins. Current
approaches to mitochondrial proteomics include the creation
of detailed catalogues of the protein components in a single
sample or the identification of differentially expressed proteins
in diseased or physiologically altered samples versus a reference
control. It is clear that for any proteomics approach prefractionation
of complex protein mixtures is essential to facilitate the identification
of low-abundance proteins because the dynamic range of protein
abundance within cells has been estimated to be as high as 10
7.
The opportunities for identification of proteins directly involved
in diseases associated with or caused by mitochondrial dysfunction
are compelling. Future efforts will focus on linking genomic
array information to actual protein levels in mitochondria.
Key Words: proteomics two-dimensional gel electrophoresis mitochondria reactive oxygen species mass spectrometry
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Introduction
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It has been estimated that there are approximately one thousand
or so different polypeptides in the mitochondrion.
1 The identification
of a majority or all of these polypeptides would be invaluable
because mitochondria play crucial and diverse roles in many
cellular processes and diseases.
18 Mitochondria are a
major source and target of free radicals,
810 and the
collapse of the mitochondrial transmembrane potential can initiate
the signaling cascades involved in programmed cell death or
apoptosis.
1116 Mitochondria play a crucial role in the
regeneration of antioxidants through the production of reducing
equivalents.
1722 Mitochondria are responsible for the
vast majority of ATP production within most cells and higher
organisms.
1 Other important roles for mitochondria include the
modulation of intracellular calcium homeostasis and thus a major
role in cell signaling, as well as biosynthesis (eg, heme) and
degradation (eg, urea cycle).
2328 Mitochondrial mutations
and dysfunction have been implicated in numerous diseases including
heart disease, myocardial preconditioning, and ischemia/reperfusion,
neurological diseases, such as Parkinsons, Alzheimers,
and Huntingtons, aging, cancer, and various neuromuscular
syndromes.
5,10,2932 The rapidly emerging field of proteomics
can provide a powerful suite of techniques for the identification
and characterization of mitochondrial proteins involved in these
processes.
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Sample Complexity Reduction: The Key to Proteomic Analysis
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The analysis and characterization of complex mixtures of proteins
are the central aims of proteomics. Whether the goal is creation
of a detailed inventory of protein components in a single sample
or identification of differently expressed proteins in disease
samples versus controls, accurate resolution of thousands of
proteins and their posttranslationally modified isoforms is
an absolute requirement.
To date, two-dimensional (2-D) gel electrophoresis is the preferred method to resolve and array authentic proteins from cellular mixtures. Combined with high-throughput mass spectrometry (MS) techniques, 2-D gels allow the simultaneous analysis of thousands of protein species. For detailed descriptions of mass spectrometry methods applicable to proteomics, see previously published review articles.33,34 Peptide mass mapping using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry has become the preferred high-throughput technique for initial identification of protein spots from 2-D gels.3539 Protein identifications made from peptide mass fingerprints can be confirmed using post source decay (PSD) MALDI-MS or tandem mass spectrometry (LC-MS/MS) fragmentation of individual peptides.34 The fragment ion spectra generated by these techniques can be used to search sequence and expressed sequence tag (EST) databases. An example of a typical MALDI-MS peptide profile and PSD-MALDI-MS spectrum generated in the laboratory of one of the authors (M.F.L.) from an in-gel digest of mitochondrial ATP synthase is shown in Figure 1. If the peptide mass fingerprints do not match any protein fingerprints present in the public databases, de novo amino acid sequence data can also be generated by fragmentation of individual peptides using LC-MS/MS or PSD-MALDI-MS.

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Figure 1. MALDI-TOF and MALDI-PSD-MS spectra of ATP synthase isolated from a 2-D gel of mouse brain mitochondria. A, Peptide mass profile. B, MALDI-PSD-MS spectrum, 1407.208 parent ion. Mitochondria were isolated as described in Melov et al.9 Proteins were prepared, run on 2-D gels, and stained with colloidal Coomassie Blue as described in Pluskal et al.129 Spots was excised, trypsin-digested, and subjected to mass spectrometry and identified by peptide mass mapping and fragment ion searching of PSD-generated spectra as described in Pluskal et al.129
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As an alternative to the 2-D gel/mass spectrometry approach described earlier, methods for the characterization of protein mixtures independent of 2-D gels have been developed in recent years. The impetus for alternatives to gels has been the perceived need for higher throughput and methods that may be easily automated.4046 In addition, questions about the limitations of 2-D gels with respect to the resolution of low-abundance or hydrophobic proteins have prompted the search for methods that are unbiased in these respects,47,48 although recent reports have demonstrated that these perceived limitations can be overcome.49
The most recent estimates place the number of genes in the human genome at approximately 40 000, far less than had previously been predicted.50 Even with this revised estimate of a "low" number of genes, the number of proteins encoded by the genome will still be far too many to see on a single gel. Current high-resolution techniques can resolve on the order of 1000 to 3000 different polypeptide spots on a 2-D gel. Consequently, some reduction in the complexity of samples is needed to display the majority of cellular proteins on gels.
Approaches to protein fractionation that focus on the solubilization and fractionation of hydrophobic and membrane proteins are especially valuable for proteomic studies of mitochondria. Many mitochondrial proteins have a basic pI, and a large number are low molecular weight. Fractionation approaches that specifically address these properties will be most useful for identifying and characterizing mitochondrial proteins. The following sections describe gel-based and gel-free approaches to the simplification of protein mixtures for analysis by mass spectrometry. Many of these methods are well suited to the study of mitochondrial proteins.
2-D Gels Are High-Resolution Protein Arrays
Two-dimensional gels are a classic technology that has enjoyed a renaissance in the past 10 years. Because of the highly parallel nature of the technique, hundreds to thousands of proteins can be visualized simultaneously.5153 Therefore, 2-D electrophoresis is an ideal tool for the resolution of complex protein mixtures. When coupled with mass spectrometry, individual polypeptide components can be accurately identified.3339 A 2-D gel is also an array of authentic proteins, ie, charge, molecular weight, and posttranslational modifications are preserved during electrophoresis. This allows direct analysis of protein isoforms that may be involved in particular metabolic or disease processes. In addition, the quantitative differences between proteins in mixtures can be determined from 2-D gel images. This allows the direct detection of differentially expressed gene products. The importance of measuring protein levels has become increasingly clear as several reports have shown that transcriptional profiles do not necessarily parallel the actual protein levels in cells5456 and cannot be used to predict posttranslational modification of proteins. Therefore, differentially expressed protein identification may turn out to be the most straightforward route to identification of drug and diagnostic targets.
There has been some debate regarding the perceived limitations of 2-D gels in the detection of low-abundance or very hydrophobic proteins.34 However, evidence presented in a recent study demonstrates that sample fractionation in combination with the correct solubilization reagents can overcome the bias toward high-abundance, cytosolic proteins49 and reveals the presence of hydrophobic and basic membrane proteins. In addition, high-percentage acrylamide gels can facilitate identification of low molecular weight proteins. Therefore, a 2-D gel-based approach is a feasible one for studying the relatively limited subset of proteins found in and associated with mitochondria.
Membrane and Hydrophobic Protein Solubilization for 2-D Gels
Membrane proteins have always proven difficult to resolve on 2-D gels.57 Several reports have described improved solubilization protocols using sequential extraction, novel detergents, and fractionation protocols for the enrichment of hydrophobic proteins.49,5759 Using these techniques, the authors resolved integral membrane proteins from yeast and Escherichia coli samples.49 These novel methods are clearly applicable to the solubilization and resolution of difficult mitochondrial membrane proteins.
High-Sensitivity Stains for 2-D Gels
Currently, there are no stains that can span the approximately 7 or 8 orders of magnitude dynamic range of cellular proteins.60,61 However, the development of high-sensitivity, mass spectrometry "friendly" stains has improved the probability of detecting and identifying low-abundance proteins in 2-D gels.62 A recent development, SYPRO Ruby protein stain is a fluorescent ruthenium-based stain that binds noncovalently to proteins in gels (Molecular Probes).63 This stain can detect low nanogram amounts of proteins in gels, is very compatible with in-gel digests for mass spectrometry, and has a linear dynamic range of 2 to 2000 nanograms, spanning the ranges of both Coomassie and silver stains.6368 An advantage of the extended dynamic range is a potentially increased accuracy of quantitative comparisons in differential display experiments.
Subcellular Fractionation
Subcellular fractionation is a useful technique for isolating proteins associated with organelles such as mitochondria. Researchers have used centrifugation or other fractionation techniques to isolate nuclei, and as a result nuclear matrix proteins,6668 as well as proteins associated with a variety of other cellular organelles or compartments including mammalian lysosomes,69 endosomes,70,71 rough endoplasmic reticulum (ER), plasma and mitochondrial membrane,72,73 mitochondrial ribosomes,74 centrosomes,75 and cyanobacterial thylakoid membranes.76 A pertinent question to any investigation of the proteomics of subcellular fractions is a discussion of the purity of the fraction. For example, mitochondrial preparations can be contaminated with cytosolic, microsomal, and ER proteins.72 It is not even clear how closely these proteins are associated in vivo and whether the connection is biologically relevant. Because the contaminating proteins will be identified using mass spectrometric approaches, the key is consistent preparation of fractions. This will prevent erroneous assumptions when different fractions are compared.
Charge Fractionation
Protein mixtures can be conveniently fractionated by charge by using narrow-range pH gradients during isoelectric focusing7780 or by using a combination of instrumentation and narrow-range pH gradients.8183 These strategies result in a series of overlapping protein patterns created with very narrow-range pH separations. This approach is well suited to the study of mitochondrial proteins because narrow-range, basic pH gradients can resolve many membrane proteins. Traditional liquid chromatography techniques can also be used to fractionate protein mixtures by charge before separation on 2-D gels. A disadvantage is that these methods require greater amounts of sample. An example of this approach is the separation of E coli proteins using anion exchange chromatography with the subsequent resolution of each fraction on 2-D gels.84
Affinity Fractionation
Affinity ligands can be useful for fractionating protein mixtures before 2-D gel separation. Affinity separations can be used to remove abundant proteins such as in the removal of albumin or IgG from serum or plasma with Cibachron Blue resins85,86 or other techniques.87 Conversely, affinity ligands can be used to enrich desirable or low-abundance proteins in a mixture using heparin,88,89 hydroxyapatite,90 various lectins, among them concanavalin A,91,92 immobilized metal-ion affinity chromatography (IMAC) resins,93 and other ligands.9498 The IMAC approach has successfully been used to enrich for calcium-binding proteins from mitochondria.93 An extension of this is to use an individual member of a protein complex as an affinity ligand or "bait" to "fish" for the rest of the proteins in the complex. Characterization of actin-binding proteins from muscle99 and the purification and identification of proteasomes100 are examples of this type of approach.
Other Fractionation Techniques
Other techniques for protein enrichment or fractionation before 2-D gels include membrane protein enrichment by differential detergent fractionation or sodium chloride treatment,101104 immunoprecipitation,105 or size fractionation,106,107 which can isolate specific protein groups.
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Liquid Chromatography and Protein Chip-Based Methods
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Numerous efforts have been undertaken to develop proteome analysis
protocols that do not require the running of 2-D gels. Several
of these approaches have the advantage that they are potentially
very high-throughput, although to date none have demonstrated
resolution of complex mixtures equivalent to that which can
be achieved in 2-D gels.
Multidimensional Liquid Chromatography
Initial efforts to characterize proteins in mixtures without using 2-D gels as a first step have focused on multidimensional liquid chromatography to decrease sample complexity coupled with mass spectrometry for protein identification.108,4042,4546 These approaches have all suffered from the disadvantage that they are not quantitative, hence not useful for differential display analyses. In addition, most of these approaches fall short of the resolution achieved with 2-D gels and are still biased with respect to pI and molecular weight. This is because very acidic proteins will be underrepresented in the mixture due to fewer tryptic cleavage sites, and low molecular weight proteins will be underrepresented due to the production of fewer peptides.42 A clear advantage is that most of these methods are very automation-friendly.
Affinity-Tagged Peptides
A refinement of the multidimensional liquid chromatography approach involves the biotinylation and affinity capture of cysteinyl peptides in an effort to minimize the bias toward larger proteins with more numerous peptides.110,111 Drawbacks to this approach are that it is not quantitative and that the purified peptides do not all ionize and fragment equally well in the mass spectrometer, leading to underrepresentation of these proteins in the analysis.110
Recently, a method was developed that takes these approaches one step further and allows quantitative analysis of protein mixtures.111 With this technique, sample protein mixtures to be compared are affinity-tagged with isotopically coded linkers or ICAT. The protein mixtures are then subjected to trypsin digestion and the tagged peptides are isolated by affinity chromatography and then analyzed by mass spectrometry. Isotopically tagged light and heavy forms of identical peptides are quantified by measuring the relative signal intensities of each pair. The relative advantages and disadvantages of this approach can be summarized in several points.
The ICAT technique has become very popular because it theoretically allows high-throughput quantitative protein identification without using 2-D gels. This can be a tremendous time-saver and eliminates labor-intensive steps.
A serious caveat is that only a small proportion of peptides contain cysteine so the technique is biased toward those proteins with several or more (relatively rare) cysteine residues. A significant number of proteins contain no cysteine residues at all, and such proteins will not be detectable by this technology. In addition, the dynamic range of the technique can be limited, depending on the mass spectrometer used.111 Unfortunately, the technique is also not truly quantitative in that the chromatographic fractionation of deuterated and nondeuterated peptides is subject to measurement errors of up to 500%. This is due to the fact that the differentially tagged peptides do not co-elute exactly.112 Finally, posttranslational modifications cannot be observed with this approach.
More recently, a method for differential stable isotope labeling of peptides using per-methyl esterification of carboxylic acids such as are present in aspartic and glutamic acids and in the carboxy termini has been developed.113 This technique should allow less biased labeling of peptides for identification by mass spectrometry. Whether the resolution and dynamic range of this newer isotope-coded affinity tagging technology for proteomics will be equivalent or better than that achieved with 2-D gels/mass spectrometry remains to be seen. However, this technique will most likely still be subject to the potential errors in quantitation observed with ICAT.
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Protein Chips or Microarrays
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The advent and success of genomic microarrays for high-throughput
analysis of transcriptional profiles have prompted analogous
approaches to proteomic analysis. In the past few years, numerous
articles have been published detailing different approaches
to protein chip technology. Most of these studies involve the
application of proteins or antibodies in arrays on glass slides
or membranes.
114119 The technology is still in its infancy
but has the potential to allow the analysis and characterization
of thousands of proteins and their interactions in a single
experiment. This would be a tremendous advantage for simultaneously
screening large numbers of samples. The drawbacks of current
methods are that posttranslational modifications, which are
known to be instrumental in the development of many diseases,
will be difficult or impossible to track by these techniques.
In an innovative departure from the traditional concept of protein
chips, some researchers are implementing the microfluidic printing
of arrayed chemistries on individual protein spots blotted onto
membranes.
49 This technique will effectively allow each spot
on a 2-D gel blot to act as a protein chip.
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Building Mitochondrial Databases
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The literature on the proteomics of mitochondria is sparse.
Few studies have been published to date, but this is bound to
change quickly as intense interest is focused on the area of
oxidative stress, disease, and mitochondrial proteins. The initial
reports have been focused on building databases or maps of mitochondrial
proteins from 2-D arrays.
72,93,109,120 This approach is driven
by the concept that detailed maps can serve as a reference for
studies of mitochondrial dysfunction and disease. Rabilloud
et al
72 created a database of 46 human placental proteins identified
from 2-D gels by mass fingerprinting with MALDI-TOF complemented
by protein sequencing and immunodetection. A limitation of this
study was the contamination of the placental mitochondrial preparation
by secreted proteins such as choriomammotropin that are present
in granules approximately the same size and density as mitochondria
and that co-purified during the subcellular fractionation procedure.
More recently, the same group has focused on identifying the
antioxidant defense system in rat mitochondria.
121 Lopez et
al
93 created a database of approximately 70 rat liver mitochondrial
proteins using a combination of affinity fractionation, 2-D
gels, and mass spectrometry. Most recently, Scheffler et al
120 identified approximately 60 proteins in mitochondria isolated
from cultured neuroblastoma cells using 2-D gels and mass spectrometry.
The three studies cited above shared the limitation that the
proteins that were resolved on broad-range pI 2-D gels were
analyzed. This meant that in most cases, the protein pI was
limited to a range of 3 to 10, and the molecular weight was
limited to approximately a range of 200 to 10 kDa. This necessarily
excludes various mitochondrial proteins including those that
are more basic. Mitochondria also contain many low molecular
weight proteins that are underrepresented in these studies.
On the other hand, because it has been estimated that the number
of different polypeptides found in mitochondria is only on the
order of one thousand,
1 there should theoretically be no problem
resolving the majority of mitochondrial proteins on a single
2-D gel. This supports the general approach of combining 2-D
gels with other techniques such as charge and affinity fractionation
to enhance resolution and therefore allow the visualization
of most of the mitochondrial proteome.
Using an alternative approach, Patterson et al109 identified approximately 100 proteins from a combination of protein databases and expressed sequence tags. In this study, purified mouse liver mitochondria were treated with atractyloside to cause opening of the permeability transition pore complex; the mitochondria were subsequently digested with trypsin and the cysteinyl peptides were purified by affinity capture. The purified peptides were analyzed by data-dependent LC-MS/MS. However, a number of factors limit this study including the fact that all peptides do not ionize with identical efficiency and thus the study will be biased toward those that do. Also, the LC-MS/MS approach is not quantitative, so the relative abundances of the represented proteins cannot be determined.
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Differential Display Analysis of Mitochondrial 2-D Gel Protein Arrays for Target Identification
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A direct approach to drug and diagnostic marker detection is
to compare the protein patterns in disease and control samples.
To this end, several differential display studies on the proteomics
of mitochondrial dysfunction have been recently published. Landin
et al
122 investigated the perturbation of mitochondria after
overdose with acetaminophen. By use of a combination of 2-D
gels, mass spectrometry, and immunodetection, a mitochondrial
acetaminophen-binding protein was identified as aldehyde dehydrogenase.
More recently, changes in mouse heart mitochondrial proteins
from transgenic mice deficient in creatine kinase were reported.
123 The authors reported several clear changes in the protein patterns
from transgenic and wild-type mice including a decrease in levels
of the mitochondrial proteins ScMit-CK and aconitase. This study
demonstrates the advantage of using a proteomic approach for
the identification of protein changes associated with transgenic
mouse models because the use of genomic methods for this purpose
remains time-consuming and indirect.
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Mitochondrial Dysfunction, Diseases, and Opportunities for Proteomic Analysis
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Mitochondrial mutations and dysfunction have been implicated
in numerous diseases, pathologies, aging, and tumorigenicity.
17,10,2932 Mitochondrial DNA deletions or point mutations that cause a
reduced capacity for oxidative phosphorylation result in a number
of specific syndromes most often including neuromuscular dysfunction.
Mitochondria play a critical role in the health and survival
of cells by providing the energy that fuels the maintenance,
repair, and turnover of cellular components. Deterioration of
mitochondrial function is therefore increasingly thought to
play a major role in aging and neurological diseases as a result
of the buildup of damage caused by reactive oxygen species (ROS)
produced by the mitochondrion itself during oxidative phosphorylation.
810 ROS production is a function both of the inefficiency of transfer
of electrons through the respiratory chain and the level of
antioxidant defenses in the cell.
8
The availability of appropriate animal models has now set the stage and facilitated the in-depth study of the proteomics associated with these processes. Homologous recombination has been used to generate several different mouse models of mitochondrial disease.8,9,124 These models fall into three separate categories: ROS toxicity, ATP deficiency, and mtDNA depletion/transcription deficiency.8 We will now briefly discuss proteomic analysis of mitochondria isolated from a model of ROS toxicity.
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Differential Protein Display of Mouse Heart Mitochondrial Proteins
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Some preliminary comparisons of mouse heart mitochondrial proteins
carried out by the authors illustrate the effectiveness of using
the 2-D gel approach for protein differential display studies
before identification of potential targets with mass spectrometry.
Mitochondrial superoxide dismutase is a protein responsible
for detoxifying superoxide produced within the mitochondria
during normal respiration.
1 Mice have been created that lack
this protein (SOD2 nullizygous mice).
125 The resulting phenotype
includes a dilated cardiomyopathy and mural thrombi that develops
within 3 days of birth.
125 The cardiomyopathy can be rescued
with antioxidant treatment, which also uncovers a severe spongiform
encephalopathy,
126 which can also be rescued on appropriate
antioxidant treatment in a dose-dependent fashion.
124 Figure 2 shows images of Coomassie Blue-stained 2-D gels of mitochondrial
proteins isolated from wild-type (WT) and SOD2 nullizygous or
knockout (KO) mice.
124 The arrows within the boxed areas indicate
a number of protein differences, both qualitative and quantitative.
Each of these polypeptides is present in sufficient quantity
for excision and processing by mass spectrometry and identification
is currently underway.
Figure 3 demonstrates the reproducibility
of the 2-D technique when applied to isolated mitochondria from
separate individuals. Each of the silver-stained gel images
is the result of a single mitochondrial preparation from an
individual mouse neonatal heart. The arrows in the four duplicate
images of each type clearly indicate a polypeptide absent in
KO that is present in WT.

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Figure 2. Two-dimensional gel images of mitochondrial proteins isolated from mouse hearts. KO indicates SOD2 knockout9; WT, wild type. Mitochondria were isolated as described in Melov et al.9 Proteins were prepared, run on 2-D gels, and stained with colloidal Coomassie Blue as described in Pluskal et al.129 Acidic proteins are to the left, basic proteins to the right, higher molecular weight at the top, and lower molecular weight at the bottom. Boxed areas are enlarged at right; arrows indicate polypeptide differences between samples.
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Figure 3. Enlarged areas from 2-D gel images of mouse heart mitochondrial proteins. KO indicates SOD2 knockout125; WT, wild type. Mitochondria were isolated as described in Melov et al.9 Proteins were prepared, run on 2-D gels, and stained with silver stain as described in Pluskal et al.129 Acidic proteins are to the left, basic proteins to the right, higher molecular weight at the top, and lower molecular weight at the bottom. Each panel shows duplicate gels from each treatment. Each gel was the result of a single mitochondrial preparation isolated from a single mouse neonatal heart. Arrows indicate a polypeptide present in WT but absent in KO.
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Future Directions
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It is clear that the science of mitochondrial proteomics is
currently a nascent but rapidly growing field. The opportunities
for identification of proteins directly involved in diseases
caused by mitochondrial dysfunction are clear and compelling.
A future objective is, without a doubt, to link the copious
amount of genomic array information that is being generated
daily to actual protein levels in mitochondria.
127,128 The synergies
between these types of data are a clear result of their complementarities.
Transcriptional profiles can identify groups of genes that are
induced or repressed with respect to a treatment or disease,
but only direct identification of the protein isoforms in a
given physiological condition can decipher the potential roles
played by posttranslational modifications.
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Acknowledgments
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This work was partially supported by NIH Grant AG18679 awarded
to S. Melov. All procedures with animals were carried out under
approved Institutional Animal Use and Care Committee protocols
of Buck Institute.
Received October 22, 2001;
revision received January 14, 2002;
accepted January 14, 2002.
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