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8/18/2019 Protein Carbonylation and Metal-catalyzed Protein Oxidation in a Cellular Perspective
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Review
Protein carbonylation and metal-catalyzed protein oxidation in
a cellular perspective
Ian M. Møller a,⁎ , Adelina Rogowska-Wrzesinskab, R.S.P. Raoc
aDepartment of Genetics and Biotechnology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, DenmarkbDepartment of Biochemistry and Molecular Biology, University of Southern Danmark, Campusvej 55, DK-5230 Odense M, Denmarkc
CH20, 3rd cross, 7th main, Saraswathipuram, Mysore 570009, India
A R T I C L E I N F O A B S T R A C T
Article history:
Received 16 February 2011
Accepted 3 May 2011
Available online 11 May 2011
Proteins can become oxidatively modified in many different ways, either by direct oxidation
of amino acid side chains and protein backbone or indirectly by conjugation with oxidation
products of polyunsaturated fatty acids and carbohydrates. While reversible oxidative
modifications are thought to be relevant in physiological processes, irreversible oxidative
modifications are known to contribute to cellular damage and disease. The most well-
studied irreversible protein oxidation is carbonylation. In this work we first examine how
protein carbonylation occurs via metal-catalyzed oxidation (MCO) in vivo and in vitro with
an emphasis on cellular metal ion homeostasis and metal binding. We then review
proteomic methods currently used for identifying carbonylated proteins and their sites of modification. Finally, we discuss the identified carbonylated proteins and the pattern of
carbonylation sites in relation to cellular metabolism using the mitochondrion as a case
story.
© 2011 Elsevier B.V. All rights reserved.
Keywords:
Carbonylation
Frataxin
Metal binding
Metal-catalyzed oxidation
Mitochondria
Reactive oxygen species
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2229
2. Protein carbonylation and metal-catalyzed oxidation in vivo and in vitro . . . . . . . . . . . . . . . . . . . . . . . 2229
2.1. What causes protein carbonylation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2229
2.2. Metal ions in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2230
2.3. Metal binding in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22302.4. MCO in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2231
2.5. MCO in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2231
J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 2 2 2 8 – 2 2 4 2
Abbreviations: 4-HNE, 4-hydroxynonenal; AGE, advanced glycation end products; ALE, advanced lipoxidation end products; CML, N(6)-carboxymethyllysine; DNP, 2,4-dinitrophenyl;DNPH, 2,4-dinitrophenylhydrazine; HICAT, hydrazide-functionalized isotope-coded affinitytag; MCO, metal-catalyzed oxidation; MRM, multiple reaction monitoring; O-ECAT, oxidation-dependent carbonyl-specific element-codedaffinity mass tag; PIC, phenyl isocyanate; PUFA, polyunsaturated fatty acid; SCX, strong cationexchange; SPH, solid phase hydrazide; SRM,single reaction monitoring.
⁎ Corresponding author. Tel.: +45 8999 3633, +45 2087 2100 (mobile).E-mail address: [email protected] (I.M. Møller).
1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.jprot.2011.05.004
a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m
w w w . e l s e v i e r . c o m / l o c a t e / j p r o t
http://dx.doi.org/10.1016/j.jprot.2011.05.004http://dx.doi.org/10.1016/j.jprot.2011.05.004http://dx.doi.org/10.1016/j.jprot.2011.05.004mailto:[email protected]://dx.doi.org/10.1016/j.jprot.2011.05.004http://dx.doi.org/10.1016/j.jprot.2011.05.004mailto:[email protected]://dx.doi.org/10.1016/j.jprot.2011.05.004
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3. Proteomic methods for the detection and quantification of protein carbonylation . . . . . . . . . . . . . . . . . . . 2231
3.1. 2D gel based proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2232
3.2. Identification of carbonylation sites in carbonylated proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 2232
3.3. Affinity enrichment based high throughput mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . 2235
3.4. Mass spectrometry-based quantitation of carbonylated residues. . . . . . . . . . . . . . . . . . . . . . . . . 2236
4. Carbonylated sites identified — MCO specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2236
5. Biological implications of protein carbonylation — the mitochondrion as a case story. . . . . . . . . . . . . . . . . 2237
5.1. Carbonylated mitochondrial proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22375.2. Carbonylation via conjugation with oxidized carbohydrates and fatty acids . . . . . . . . . . . . . . . . . . 2238
5.3. Protein turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2238
5.4. Intracellular signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2239
6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2239
A c k n o w l e d g e m e n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 3 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2239
1. Introduction
The production of Reactive Oxygen Species (ROS) is anunavoidable consequence of aerobic metabolism. In mamma-
lian cells, the majority of ROS production is localised to
mitochondria. Also in non-photosynthesizing plant cells
mitochondria are the main source of ROS. In contrast, in
photosynthesizing plant cells, it is the chloroplasts and the
peroxisomes that are by far the dominant ROS sources [1]. ROS
can oxidize DNA, carbohydrates, unsaturated fatty acids and
proteins. Proteins can be oxidized in many different ways.
While some of these oxidations are reversible, e.g., disulphide
formation, which plays a role in metabolic regulation, others
are not and can lead to inactivation of the modified protein.
The cell therefore has developed ways to minimize ROS
production, remove ROS once formed and repair at leastsome of the damage [2,3]. At the same time ROS can act as
messenger. Under stress such as disease, pathogen attack,
nutrient deficiency, and toxicity, ROS production and concom-
itant damage generally increases. When the cellular defence
mechanisms are overwhelmed, the amount of damage includ-
ing that of protein oxidation accumulates and this can lead to
cell death. Understanding protein oxidation is therefore an
important part of understanding cellular stress response.
Protein carbonylation is the most common and best
studied irreversible protein oxidation and we will here first
describe what causes protein carbonylation. We will then
briefly describe proteomic methods for identifying carbony-
lated proteins and their sites of modification. In the finalsections, we will review the consequences of protein carbon-
ylation for the function of the cells and organisms. Through-
out this review, case stories will be selected from amongst
mitochondrial proteins.
2. Protein carbonylation and metal-catalyzedoxidation in vivo and in vitro
2.1. What causes protein carbonylation?
Carbonyl groups (reactive aldehydes and ketones) can be
introduced into proteins by oxidation of amino acid side
chains or they can be generated through oxidative cleavage of
proteins by α-amidation pathway or by oxidation of glutamyl
side chains, leading to formation of peptides with an α-ketoderivative at the N-terminal. Protein carbonylation can also be
generated by conjugation with aldehydes produced during
peroxidation of polyunsaturated fatty acids (PUFA) (so-called
Advanced Peroxidation End Products, ALE) and with reactive
carbonyl derivatives generated by reaction with reducing
carbohydrates (so-called Advanced Glycation End Products,
AGE) (e.g., [4]).
Reactive oxygen species (ROS) that lead to protein oxida-
tion can be generated via a number of physiological and non-
physiological processes, primarily as by-products of normal
mitochondrial metabolism. For experimental purposes differ-
ent methods can be employed in the generation of ROS, for
example X-ray or UV irradiation or chemicals. Fortunately,living organisms are rarely exposed to that type of damage.
The most common mechanism of protein carbonylation in
living cells appears to be metal-catalyzed oxidation (MCO).
MCO typically occurs when reduced metal ions like Fe2+ orCu+
interact with H2O2 in the so-called Fenton reaction and
produces the extremely reactive hydroxyl radicals [5]:
Fe2þ þ H2O2 → Fe3þ þ HO þ HO• ð1Þ ðFenton reactionÞ
The hydroxyl radical oxidizes amino acid side chains or
causes protein backbone cleavage both resulting in the
formation of carbonyl groups. It has been argued that in
bacteria MCO is the only source of protein carbonylation [6].
It is becoming evident that there exists a close interplay
between the different types of protein carbonylation and MCO,
but the physiological mechanisms controlling these processes
are not yet completely understood. It has been shown that
MCO and free radicals play a major role in the formation of
AGEs and AGE-induced protein cross-linking [7,8]. And MCOof
PUFA in the presence of proteins can also lead to formation of
N(6)-carboxymethyllysine (CML) by reaction of the PUFA
breakdown product, glyoxal, with the lysine side-chain. This
suggests that lipid oxidation plays a role in AGE formation as
well [9]. Dihydroxyacetone phosphate, a glycolytic intermedi-
ate, spontaneously decomposes to methylglyoxal, which can
also react with protein amino acid to give AGE products [10].
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This is an example of protein carbonylation formed in the
absence of MCO.
2.2. Metal ions in vivo
To get an idea of the importance and mechanism of MCO, let
us first briefly consider the amounts of metal ions present in
livingorganisms. We will here concentrate on two of themetalions catalysing the Fenton reaction, Fe and Cu (Table 1).
The average total concentration of Fe and Cu ions in
healthy living cells is generally in the micromolar range
although specialized cells like the erythrocytes with their
massive haemoglobin content may contain as much as 20 mM
Fe. In contrast, the concentration of free ions is orders of
magnitude lower. In fact, with an intracellular concentration
of 10−18 M, a yeast cell contains not a single free Cu ion! [11].
The concentration of total and free or “labile” Fe ions appears
to be highly variable between different cell types (Table 1). At
least for the concentration of “labile” Fe ions, this is “a
methodically defined quantity” (see [12] for a discussion), but
the concentration appears to increase in diseased humans
[13].
In mitochondria about 75% of the Cu and Fe ions are
associated with the membrane fraction (containing mainly
the inner membrane), which is consistent with the fact that
the electron transport chain contains a number of abundant
metal-binding proteins [14].
2.3. Metal binding in vivo
The use of ferric and ferrous ions in living cells developed in
the absence of molecular oxygen in the atmosphere. With the
advent of oxygen-evolving photosynthesis the concentration
of oxygen in the atmosphere rose and it became necessary to
prevent the damaging Fenton reaction (e.g., [15]). Eukaryotic
cells contain hundreds of metalloproteins that require iron
and/or copper ions to carry out their function many of which
are found inside organelles such as the mitochondria [16].
These metalloproteins include metal ion transporters, pro-
teins stabilized by metal binding and enzymes that use the
metal ions in the catalytic site. From the moment the ion is
recognized outside the cell it is bound by dedicated metal-
binding proteins and handed down a bucket-brigade of other
such proteins and transporters until it reaches its final
destination. In this way the cell can regulate the metal ion
supply while at the same time keeping the concentration of
free metal ions extremely low. This will minimize metal ion
binding to undesirable proteins and limit the concomitant risk
of oxidative damage due to the interaction with O2 and H2O2[17].
Cu and Fe ions are bound to proteins by the side chains of 4–6 amino acid residues, mainly Cys, Met, His, Glu, Asp or Tyr
[18]. The total relative abundance of these amino acids in
proteins from eukaryotes is 21% (Uniprot database), which
means that there is a very large number of potential metal
binding sites in a cell, most of which are rarely or never
occupied. The ligands are often found on neighbouring
helices, which means that it is virtually impossible to predict
metal binding to proteins especially where the secondary and
tertiary structure is unknown [16].
To obtain the right picture we need to compare the
total concentration of Fe and Cu ions, which is normally in
the micromolar range (Table 1), to the total protein concen-
tration in a selected cellular compartment. For instance, in
the mitochondrial matrix the protein concentration is
>500 mg/ml [19]. Assuming that the proteins are 50 kDa on
the average, this is equivalent to >10 mM of proteins. Each of
these protein molecules may contain one to several potential
metal binding sites, most of which are quite unspecific. Thus,
the number of potential metal binding sites on proteins
exceeds the total number of metal ions by several orders of
magnitude.
Not all bound Fe and Cu ions can catalyze the Fenton
reaction. If all the coordination sites of the metal ion are
occupied by ligands then binding of oxygen or ROS is not
possible. This is what happens when EDTA binds one of these
ions, which is used to terminate in vitro MCO (see below). In
fact, the inclusion of EDTA in the homogenization medium
when making extracts of biological tissues is recommended to
prevent Fenton reactions to take place during sample han-
dling. On the other hand, oxygen or ROS binding to a protein-
bound metal ion is not a guarantee of the Fenton reaction. If
the metal ion is strongly bound with slow on-off rates the
oxygen intermediate will be stable and will not give rise to the
hydroxyl radical. This type of mechanism is observed in
enzymes like catalase, superoxide dismutase (SOD) and
Table 1 –
Concentration of metal ions in living cells.
Cell/tissue Total iron Free iron Total copper Free copper Reference
Mammalian cells 0.001–10 μM – 68 μM – [17]
Rat plasma 0.25–3 μM – 0.26–2.9 μM – [112]
Rat hepatocytes 1000 μM 9.8 μM – – [113]
Human cell lines – 0.2–1.5 μM – – [114]
Human plasma – Undetectable a
0.5–5.8 μM b– – [13]
Yeast cells – – 170 μM
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cytochrome c oxidase, whichall interact with eitherH2O2 or O2(e.g., [20]).
Fe ions are sequestered in several cellular compartments
by ferritins, a class of proteins that store the metal ions in a
safe and easily accessible form [21,22]. In mammalian cells
ferritins are found in the cytosol, nucleus and mitochondria,
while in plant cells they are also found in plastids. Knock-out
mutants of ferritins typically give rise to cells that areoxidatively stressed although the presence of several ferritin
isoforms can mask that effect [23].
Frataxin is another protein contributing to Fe homeostasis
specifically in the mitochondria where it is involved in the
biosynthesis of FeS clusters and cytochromes [24–27]. Frataxin
deficiency causes severe illness (Friedreich ataxia) or embryo
lethality in both humans and plants [25,28] and, in knock-out
mutants, the absence of frataxin induces severe oxidative
stress in mitochondria [29,30]. In contrast, overexpression of
frataxin resulted in yeast cells that were more resistant to
oxidizing agents and contained fewer carbonylated proteins
[24].
2.4. MCO in vitro
MCO has often been used in model studies as a convenient
way to generate protein carbonylation. The pure protein or
biological protein mixture is incubated at 20–30 °C for few
minutes and up to several hours with Fe3+ and ascorbate or
Cu2+ and ascorbate and the reaction is stopped by chelating
the metal ions with e.g. EDTA followed by dialysis. Typically,
100–1000 μM metal salts have been used, but their low
solubility at neutral pH would have kept the concentration
of free metal ions at a much lower level but probably orders of
magnitude above that observed in biological systems. This
would ensure binding to sites, which might not normally bind
metal ions. In some cases the metal ion concentration used
exceeded the protein concentration many-fold, so each
protein molecule would have bound several or many metal
ions mostly at lower-affinity sites. The protein modifications
(carbonylation and other oxidative modifications) subse-
quently identified have no doubt been very useful for
developing methods for studying protein oxidation, but the
sites and the type of modification found may not reflect the in
vivo response [6,31–35].
We are only aware of one study in which in vitro MCO was
conducted at a series of metal ion concentrations ranging in
stoichiometry from 1 Fe per1500 protein molecule (BSA) to 1 Fe
per 1.5 protein molecule [6]. This type of study potentially
yields much more useful information about the MCO mech-
anism (see Section 4). However, none of the above studies
have linked carbonylation with function, so we still know
relatively little about the effect of the specific modifications
observed on the structure and function of the affected
proteins.
2.5. MCO in vivo
As we have seen, the concentration of free metal ions in the
living cell is extremely low and the proteins involved in the
uptake, transport and processing of metal ions bind them in
such a way that they are unable to catalyze the Fenton
reaction. This is also true for most of the proteins using metal
ions for structure and/or function. A special case is provided
by the enzymes involved in ROS or oxygen metabolism. These
enzymes must be able to bind molecules like H2O2 without
catalyzing the Fenton reaction.
In some cases, the Fenton reaction is thought to be part of
the desired mechanism. The transcription factor PerR in
Bacillus subtilis contains two His residues coordinating boundFe2+. Upon exposure to low levels (
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product of Met) or N-formylkynurenine and kynurenine
(oxidation products of Trp) also contain aldehyde and ketone
groups, respectively. In spite of this, these products are never
mentioned in studies involving DNP-conjugation. This is an
important point that should be resolved.
The ultimate goal, to identify carbonylation type and
localise the affected residue, is technically challenging and
only recently appropriate methods have been developed andtested in vitro and in vivo [42]. In this section we will
summarize proteomic approaches used for identification
and quantitation of carbonylated proteins and give an
overview of the emerging mass spectrometry- (MS-) based
advances that allow direct identification of carbonylation sites
in the protein. Challenges that are associated with this type of
analysis will be discussed.
3.1. 2D gel based proteomics
To date the majority of proteomic studies of protein carbon-
ylation have used two-dimensional gel electrophoresis (2-DE)
coupled with MS as a means of protein separation, quantifi-
cation and identification. Supplementary Table S1 lists over 50
proteomic studies identifying carbonylated proteins using 2-
DE and carbonyl-specific detection methods. This approach
has been successfully applied for studying various diseases in
human subjects and in animal models e.g. Alzheimer disease,
systemic lupus erythematosus, chronic obstructive pulmo-
nary disease, glaucoma and diabetes. In plants it has been
used to follow protein oxidation during the plant's life cycle,
germination and O3 and CO2 stress. Using cultured cell lines,
bacteria and yeasts this method was applied to determine the
impact of various stress factors on protein carbonylation e.g.
H2O2, MCO, arsenite, X-ray irradiation and drug-induced
toxicity as well as displacement of iron and copper homeo-
stasis. It has also been used extensively to study protein
carbonylation during ageing. Recently, two new areas of
application have emerged, following changes in protein
oxidation in sentinel aquatic species and assessing the quality
of frozen stored fish meat (see supplementary Table S1 for
references).
By combining 2-DE with carbonyl-specific probes it is
possible not only to isolate and identify carbonylated proteins,
but also to quantify the degree of carbonylation of each
protein in relation to its overall quantity. Several different
chemical probes for detection of protein carbonyls have been
developed including DNPH, tritiated sodium borohydride,
biotin hydrazine-containing probes and fluorescent probes.
Properties of these probes have been reviewed recently [43].
The most widely used system for detecting carbonylated
proteins on 2D gels is based on DNPH derivatisation and
immunodetection with anti-DNPH antibody. It was first
published in 1994 by Shacter and colleagues [44] and is
commercially available under trade name OxyBlotTM. Three
variants of the protocol are used depending on when in the
process the DNPH derivatisation step is carried out. It can be
carried out before isoelectrofocusing [45]; right after isoelec-
trofocusing [46,47] or post electrophoretically [48]. Pre-elec-
trophoretic DNPH derivatisation of proteins requires low pH
and the excess reagent has to be removed, which can lead to
uncontrolled loss of proteins. DNPH derivatisation changes
protein mobility and therefore it is notpossible to compare the
patterns of carbonylated and non-carbonylated proteins
directly. Preparation of control samples by treating protein
extracts in the same way as for DNPH labelling, but without
DNPH, is mandatory. Post-electrophoretic or isoelectrophore-
tic staining overcomes those problems and allows direct
comparison between labelled and non-labelled patterns,
which facilitates the quantitation process and MS identifica-tion [46–48].
Protein carbonyls can also be detected by labelling with
fluorescent carbonyl-reactive probes, for example with fluo-
rescent hydroxylamine [49] or fluorescein-5-thiosemicarba-
zide [50]. A similar approach based on biotinhydrazide
derivatisation followed by visualisation with avidin fluores-
cein probes has also been used [51]. Immunoprecipitation of
carbonylated proteins prior to electrophoretic separation has
also been successfully applied for identification of oxidised
proteins in the matrix of rice leaf mitochondria [52] and
susceptibility of endoplasmic reticulum proteins in HL-60 cells
[53].
The advantageof 2-DE based approach is that it is relatively
easilyimplemented in a molecular biology laband MS analysis
of proteins can be carried out in a MS dedicated facility. The
separation power of 2-DE simplifies subsequent MS analysis
and the digestion of eachspoton a 2Dgel gives riseto peptides
from typically one or two proteins. Therefore subsequent
interpretation of the obtained data is relatively simpleand one
would expect that it should promote identification of modi-
fied/carbonylated peptides. That is unfortunately not the case
with the exception of oxidised Trp residues, which have been
reported in a number of studies [54–60]. However, oxidized Trp
has recently been shown to be introduced during gel
electrophoresis [61]. Until now only a single study has
identified a peptide carrying a carbonylated Arg residue in
protein extracted from a 2D gel spot [62].
3.2. Identification of carbonylation sites in carbonylated
proteins
MS is a central technology in the discovery of post-transla-
tional modifications of proteins, enabling mapping of modi-
fication sites and subsequent quantification of the abundance
of the modified peptides. It also allows detection of new types
of structures. Modifications are detected from tandem mass
spectra by observing shifts in the expected positions of
fragment peaks compared to the native peptides. In recent
years approaches dedicated to identification of several
different types of modifications (e.g. phosphorylation, glyco-
sylaton, acetylation) have been developed [63,64].
Investigation of any post-translational modification of
proteins presents immense analytical challenges (for reviews
please see [65,66]) mainly due to the fact thatthey exist in cells
at substoichiometric levels. This means that a modification of
a given site is often present in only a small fraction of the
protein molecules of a given type. This phenomenon is also
true for carbonylated proteins.
In positive-ion operating conditions, ionisation of peptides
strongly depends on the presence of basic sites. These sites
include the N-terminal amine and the side group of Lys, Arg
and His residues. Generation of carbonyl products of Arg and
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Table 2a – Single amino acids modifications induced by oxidative damage. a Atomic composition and monoisotopic mass of the difference between the native amino acid and the oxidized product are given.
Aminoacid
Product Composition Monoisotopicmass change
Reactive aldehydeor ketone group
Known to beinduced by MCO d
Modification of amino acid side chain
Ala Serine +1O +15.99492
Arg Glutamic semialdehyde −5H − 1C − 3N +1O −43.05343 √ √
Arg +14 Da b −2H +1O +13.97927 c
Arg Hydroxyarginine +1O +15.99492
Asn 3-hydroxyasparagine +1O +15.99492
Asp Decarboxylation −2H − 1C − 1O −30.01056 √
Asp 3-hydroxyaspartic acid +1O +15.99492
Cys Dehydroalanine −2H − 1S −33.98772
Cys Serine −1S +1O −15.97716
Cys Sulfenic acid +1O +15.99492
Cys Sulfinic acid +2O +31.98983
Cys Sulfonic acid +3O +47.98474
Gln +14 Da b −2H +1O +13.97927 c
Gln Hydroxyglutamine +1O +15.99492
Glu Decarboxylation −2H − 1C − 1O −30.01056 √
Glu +14 Da b −2H +1O +13.97927 c
Glu Hydroxyglutamic acid +1O +15.99492
His Aspargine −1H − 2C − 1N +1O −23.01598 √
His Aspartic acid −2H − 2C − 2N +2O −22.03197 √
His Aspartylurea −2H − 1C − 2N +2O −10.03200
His Formylaspargine −1H − 1C − 1N + 2O + 4.97900
His 2-oxohistidine +1O +15.99492
Ileu +14 Da b −2H +1O +13.97927 c
Ileu 4-hydroxyisoleucine +1O +15.99492
Leu +14 Da b −2H +1O +13.97927 c
Leu 3-hydroxyleucine +1O +15.99492
Lys Aminoadipic semialdehyde −3H − 1N +1O −1.03163 √ √
Lys +14 Da b −2H +1O +13.97927 c
Lys Hydroxylysine +1O +15.99492 √
Lys Lysine hydroperoxide +2O +31.98983
Met Aspartate semialdehyde −4H − 1C − 1S +1O −32.00846 c
Met Methionine sulfoxide +1O +15.99492
Met Methionine sulfone +2O +31.98983
Met Homocysteic acid −2H − 1C +3O +33.96910
Phe Hydroxyphenylalanine, tyrosine +1O +15.99492
Phe Dihydroxyphenylalanine (DOPA) +2O +31.98983
Phe Trihydroxyphenylalanine (TOPA) +3O +47.98475
Pro Pyrrolidinone −2H − 1C − 1O −30.01057 √
Pro Pyroglutamic acid −2H +1O +13.97927 √
Pro Glutamic semialdehyde +1O +15.99492 √ √
Pro Hydroxyproline +1O +15.99492 √
Pro Glutamic acid +2O +31.98983 √
Ser Hydroxyserine +1O +15.99492
Thr 2-amino-3-ketobutyric acid −2H −2.01560 √ √
Thr Hydroxythreonine +1O +15.99492
Trp Kynurenine −1C +1O +3.99490 c √
Trp Oxolactone −
2H +1O +13.97927Trp 2,4,5,6 and 7-hydroxytryptophan +1O +15.99492 √
Trp Hydroxykynurenine −1C +2O +19.98983 c √
Trp b-unsaturated-2,4-bis-tryptophandione −4H +2O +27.95853
Trp Tryptophandione, dihydrodioxoindole −2H +2O +29.97418
Trp N-formylkynurenine, dioxindolylalanine,
dihydroxytryptophan
+2O +31.98983 c √
Trp Hydroxy-bis-tryptophandione −4H +3O +43.95345
Trp Hydroxy-N-formylkynurenine +3O +47.98474 c
Trp Dihydroxy-N-formylkynurenine +4O +63.97966 c
Tyr Dihydroxyphenylalanine (DOPA) +1O +15.99492
Tyr Trihydroxyphenylalanine (TOPA) +2O +31.98983
Val +14 Da b −2H +1O +13.97927 c
Val Hydroxyvaline +1O +15.99492
(continued on next page)
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Lys (to give glutamic and 2-amino-adipic semialdehydes) is
associated with loss of guanidine and ammonia groups, which
carry the positive charge. This leads to a decreased ionisation
efficiency and reduces the chance of detecting those peptides
using positive-ion mode MS.
What makes analysis of oxidised proteins exceptionally
challenging is the fact that there are many types of modifica-tions of proteins that result in creation of carbonyl residues
and they come in many different sizes (Tables 2a and 2b).
Apart from those modifications there are also many other
oxidative modifications that can be introduced in different
amino acids which can co-exist in oxidised proteins together
with carbonylated residues. Table 2a compiles known prod-
ucts of single amino acid modifications induced by oxidative
damage excluding cross-linked products. Atomic composition
and monoisotopic mass of the difference between the native
amino acid and the oxidized product is given for an easier
inclusion of the modifications into MS data searches. It should
be noted that many of the single amino acid modifications
included in the list have not yet been observed in biologicalsamples. However, as proved by various experiments using
isolated amino acids and model peptides, these modifications
potentially can be induced by hydroxyl radicals and they
should therefore be included as possible products of MCO.
Additionally a number of products (Table 2a) potentially
containing carbonyl residues, but not commonly regarded as
markers of oxidative stress, is listed. These products, although
probably notabundant, may also contribute to the overall pool
of protein carbonyls in biological systems and they merit
further study.Yet another angle of complexity is the fact that two
different oxidation products can have the same mass differ-
ence. For example, Pro hydroxylation to hydroxyproline
results in addition of +16 Da. The same mass difference is
observed during the conversion of Pro to glutamic semialde-
hyde. Thus thesetwo very distinct modifications of Pro cannot
be distinguished solely by MS.
Together with the problem of the dynamic range comes also
the complexity challenge, which makes analysis of MS data
significantly more difficult. Identification of low-occupancy
sites among an excess of unmodified peptides is addressed by
techniques that rely on affinity enrichment methods described
below. However, to date no bioinformatics approaches havebeen developed to solve the complexity problem.
Typical MS data searching algorithms (e.g. Mascot) can
search with a limited number of set modifications and the
Table 2a (continued)
Aminoacid
Product Composition Monoisotopicmass change
Reactive aldehydeor ketone group
Known to beinduced by MCO d
Nitrosylation and chlorination
Phe 2-nitrophenylalanine −1H 1N +2O +44.98508
Trp 6-Nitrotryptophan −1H 1N +2O +44.98508
Tyr 3-cholorotyrosine −1H +1Cl +33.69103
Tyr 3-nitrotyrosine −1H 1N +2O +44.98508
a Compiled from [60,69,101,115–122];b Exact structure of these products is unknown;c Residues potentially containing reactive aldehydes and ketone groups;d Based on [115,122].
Table 2b – Typical advanced glycation and lipoxidation products. a Atomic composition and monoisotopic mass of thedifference between the native amino acid and the oxidized product are given.
Amino acid Product Composition Monoisotopicmass change
Reactive aldehydeor ketone group
Advanced glycation products (AGE)
Arg Glyoxal derived imidazolone +2C + 1O +39.99491 √
Arg Glyoxal derived hydroimidazolone +2H + 2C + 1O +42.01056 √ Lys Glyoxal derived carboxymethyllysine (CML) + 2H + 2C +2O +58.00548 √
Lys Methylglyoxal derived carboxyethyllysine (CEL) + 3H + 3C + 2O + 71.01331 √
Arg Methylglyoxal derived argpyrimidine +4H + 6C + 1O +92.02622 √
Arg, Lys Methylglyoxal derived tetrahydropyrimidine + 6H + 7C + 4O + 154.02661 √
Arg 3-deoxyglucosone derived imidazolone A + 8H + 6C +4O + 144.04226 √
Advanced lipoxidation products (ALE)
Arg Malondialdehyde derived N-propenalarginine + 4H + 3C +1O + 56.02622 √
Lys Malondialdehyde derived N-propenallysine + 4H + 3C +1O +56.02622 √
Lys Acrolein derived FDP-lysine +6H +6C + 1O +94.04187 √
Lys Croton aldehyde derived dimethyl-FDP-lysine + 10H + 8C + 1O + 122.07317 √
Cys, His, Lys 4-oxo-2-nonenal (Michael adduct) + 11H +9C +2O + 154.09990 √
Cys, His, Lys 4-hydroxy-2-nonenal (Michael adduct) + 16H + 9C + 2O + 156.22480 √
a
Compiled from [123–125].
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searching time dramatically increases with an increased
number of modifications. Therefore it is necessary to search
the same data separately several times [67] and all possible
combinations of modifications cannot be included. To be able
to find a modified residue using computer-aided search
algorithms (e.g. Mascot) it is required to define the mass
difference between the modified and non-modified residues.
Therefore only known modifications can be found with thisapproach. New types of modifications can only be found if
manual data analysis is carried out, butthisapproachis labour
intense and time consuming, and cannot be used effectively
with complex protein mixtures.
Identification of oxidatively modified peptides with a search
algorithm poses several other technical problems. MS-based
analysis of proteins inevitably involves the enzymatic degrada-
tion of proteins to peptides. The enzyme of choice is trypsin.
This protease has high cleavage specificity by cleaving C-
terminally to Arg or Lys residues. Oxidised Lys and Arg residues
become inaccessible to trypsin proteolysis, leading to a higher
number of missed cleavagesthannormal [68]. As a consequence
the resulting peptides might be outside the analytical range of
mass spectrometers and the possibility of a higher numbers of
missed cleavages has to be included in search algorithms
increasing the search space and search time exponentially and
causing high false discovery rates. Oxidative attack on proteins
results in protein backbone cleavage [69]. Peptides produced by
oxidative fragmentation lack the C-terminal enzyme specificity
(Lys or Arg residues for trypsin) that is required for enzyme-
specific identification. Thus search algorithms used must
include an option of searching for semitryptic or non-tryptic
peptides, which again increases search space and search time
andcan cause high false discovery rates [68]. Oxidation can also
induce protein and peptide cross-linking. Finding cross-linked
peptides in a mixture of proteins is very difficult and requires
special search algorithms.
In spite of the difficulties in identifying carbonylation sites,
the combination of the methods described above with the
enrichment methods described in the following Section 3.3
has lead to the identification of several hundred carbonylation
sites in specific proteins. This will be treated in Section 4.
3.3. Affinity enrichment based high throughput mass
spectrometry
Proteomic methods that rely exclusively on MS as a means of
protein identification, characterisation and quantitation re-
quire a pre-fractionation step that allows for the enrichment
of a certain group or type of proteins — in this case
carbonylated proteins. Some of the specific chemical probes
that have been developed for the detection of carbonyl
residues, but also new probes that have been developed, can
serve to affinity purify carbonylated proteins. The use of
different chemical probes for specific enrichment of carbony-
lated proteins has been reviewed recently [42].
Biotin-hydrazide is an aldehyde and ketone reactive probe;
it reacts with carbonyl groups to form a Schiff base, which is
then reduced with sodium cyanoborohydride to prevent
hydrolysis. The resulting biotin hydrazone can act as a
“handle” by which carbonylated proteins can be selected
with immobilized avidin or streptavidin resins. This approach
has been used in a number of proteomic studies to enrich for
carbonylated peptides and identify carbonylation sites in
model proteins [31] and in complex protein mixtures e.g.
yeast lysate [34,68,70], rat plasma [71] and human plasma [67].
An important fact is that the interaction between biotin and
avidin is very strongand therefore in most studies monomeric
avidin resins are used. Even though the interaction is so
powerful that efficient elution of peptides cannot be achieved,this approach is preferred for protein enrichment [42].
2-Iminobiotin, the cyclic guanidino analogue of biotin, first
described by Hofmann and Axelrod in 1950 [72] is an attractive
alternative to biotin. This tag has a pH-dependent interaction
with avidin. At high pH the free base of 2-iminobiotin forms a
stable and tight complex with avidin (KD=3.5×10−11 M). At low
pH, the interaction becomes much weaker (KD
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3.4. Mass spectrometry-based quantitation of
carbonylated residues
In order to achieve a comprehensive understanding of the
mechanisms governing protein oxidation in complex biolog-
ical systems we need quantitative tools to follow the oxidative
damage to proteins in time and within different cellular
compartments. While MS has been mainly used to mapcarbonylation sites within proteins, spectrophotometry and
immunochemistry combined with 2-DE have been the
methods of choice to do the quantitative measurements.
The ultimate goal of redox proteomic studies is to detect and
quantify the oxidized amino acids within proteins. Relative
quantification studies using stable isotope coding allows
comparing the degree of oxidation of a particular site between
two or more samples. Isotopomers of DNPH [78], Girard-P
reagent [32], O-ECAT [35], HICAT [79], iTRAQ [80,81], PIC
reagent [82] and most recently targeted 18O-Labeling [77]
have been used in relative quantification studies of carbony-
lated proteins. Even though relative quantitation provides
invaluable insights into the mechanisms of protein carbonyl-
ation very little is known about the fraction of any particular
protein or protein site being oxidized in selected condition.
Absolute quantitation by MS can be achieved by adding an
internal standard. Because quantitation in MS is based on
integrated ion intensities it is crucial to take into account that
MS signal intensities depend not only on the amount of
sample, but most importantly on the chemical properties of
the peptide. Therefore to measure the absolute quantity of a
peptide a synthetic heavy isotope labelled peptide of the same
sequence and modification has to be used. The method
utilizing internal standard is often referred to as single
reaction monitoring (SRM) or multiple reaction monitoring
(MRM) if several peptides are being followed in a single
experiment. In recent years this technique has been rapidly
developing and is considered one of the most effective tools
for quantitative proteomics, especially clinical proteomics
[83]. In proteomic studies it has not yet been widely used for
monitoring protein carbonylation, but successful quantifica-
tion of 4-HNE and glutathione adducts to carnosine and
anserine dipeptides in rat skeletal muscles [84] provides a
valuable proof of concept.
4. Carbonylated sitesidentified — MCO specificity
Using the methods described in Section 3, it is possible to
identify carbonylated proteins and the site of carbonylation.
We have found 12 proteomic studies in which specific
carbonylated Arg, Lys, Pro and Thr residues have been
identified in a variety of organisms although no plant study
has been found. A total of 456 non-redundant sites in 208
proteins have been identified (Supplementary Table S2). Most
of these studies were in vitro MCO where oxidation was
effected at one fixed (high) metal ion concentration or they
were in vivo MCO where samples were taken under control
conditions (e.g. healthy humans) and/or under stressed
conditions (patients or oxidatively stressed cells). The most
commonly carbonylated amino acid was Lys followed by Pro
and Arg/Thr (Table 3). In most cases there is no information
about metal ion binding and how that might contribute to the
oxidation pattern observed.
In one study, in vitro MCO was conducted at a series of
metal ion concentrations ranging in stoichiometry from 1 Fe
per 1500 protein molecule (BSA) to 1 Fe per 1.5 protein
molecule [6]. The ascorbate concentration was also varied so
that there were always 250 ascorbate molecules for every Fe3+
ion thus ensuring the production of an average of 250 HO • at
each metal-binding site. This kind of study yields more
information about the MCO mechanism.
Based on the observation of in vitro carbonylation in BSA,
Maisonneuve et al. [6] suggested that some positions in the
amino acid chain are more prone to carbonylation, so-called
hotspots. A hot spot was defined as a four-residue window
containing three Arg, Lys, Pro or Thr (RKPT) residues out of
which at least one is a Pro. Further, there should be an iron-
binding residue and a hydrophobic residue in the proximity.
Although the majority of carbonylated sites occurred in RKPT
rich regions in BSA, the hot spot rule was not very effective in
predicting carbonylations in other carbonylated proteins [6].Maisonneuve et al. [6] also identified carbonylated sites in
BSA at different MCO levels. It was observed that carbonylated
sites at higher MCO levels contained all the sites that were
carbonylated at lower levels. It was therefore proposed
that there is an order in the propagation of carbonylation in
proteins.
Finally, Maisonneuve et al. [6] made the interesting
suggestion that RKPT sites may be more susceptible to
carbonylation near (in the protein primary structure) a
carbonylated site. For instance, many of the carbonylated
sites observed at higherMCO levelswere very close to the sites
that were carbonylated at lower levels, indicating some sort of
clustering.To test whether carbonylated sites have a tendency to
cluster in the protein primary structure as suggested by
Maisonneuve et al. [6], we performed a Monte Carlo simulation
(~ random sampling) of the distance between carbonylation
sites. The pattern of simulated distance between all RKPT
sites, i.e. all potential carbonylation sites, is nearly identical to
the actual observed pattern of carbonylation (Fig. 1A). In
contrast, the simulated pattern of proportion/distance
obtained for carbonylated sites is very different from the
actual pattern (Fig. 1B). Due to the large number of available
RKPT sites, carbonylation has a wide scope to vary. Any
deviation from this (random) expected pattern is therefore
seen as a clear indication of some selective force at work.
Table 3 – MCO specificity as indicated by the number of times different amino acids have been carbonylated.
Amino acid Carbonylated frequency a
Arginine 90
Lysine 147
Proline 129
Threonine 90
Total 456
a From 208 non-redundant carbonylated proteins [6,31–35,62,67,68,
70,126,127].
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When carbonylations are assumed to fall in surface-exposed
region, the proportion at close distance is slightly higher.
When carbonylations are assumed to be in RKPT rich regions
the simulated pattern is very similar to the actual pattern
(Fig. 1B). These are just two extreme assumptions; the actual
pattern may be the cumulative result of several factors
including the presence of a metal binding site. However, the
simulation confirms the conclusion of Maisonneuve et al. [6]that protein carbonylations have a strong tendency to cluster
probably near metal binding sites. More studies of this type
should be performed to gain a better understanding of the
MCO mechanism.
5. Biological implications of proteincarbonylation — the mitochondrion as a case story
Carbonylated proteins have been identified in many different
studies in various biological systems (see Supplementary
Table S2). It would not be meaningful to discuss all of these
studies here. Instead we have decided to select the mitochon-
drion as a case story.
The mitochondrion is the main site of ROS production in
mammalian cells [37] and in non-photosynthesizing plant
cells [1,3]. Superoxide is produced at seven sites in mitochon-
dria, the two major being complexes I and III in the respiratory
chain. Most of the superoxide is released into the matrix space
where it can be converted into H2O2 by MnSOD. There are
several enzyme systems present in the mitochondrial matrix
that can remove the H2O2 [3]. H2O2 can also diffuse across the
inner mitochondrial membrane into the intermembrane
space presumably through aquaporins [85] and across the
outer membrane through porin into the cytosol and when a
“free” metal ion is available, the Fenton reaction can take
place. Although the total metal ion concentration in mito-
chondria is very high, especially in the membranes [14], the
free metal ion concentration is strictly regulated by metal-
binding proteins such as ferritin and frataxin (see Section 2.3).
That this is important is illustrated by the observation that
frataxin deficiency is the cause of Friedreich's ataxia, a humancardio- and neurodegenerative disease [25]. At the cellular
level, frataxin deficiency in yeast leads to the accumulation of
a number of carbonylated proteins many of which are
mitochondrial [24,86]. Frataxin overexpression, on the other
hand, leads to a marked decrease in the amount of carbony-
lated protein especially in the mitochondria and an increased
stress tolerance [24].
5.1. Carbonylated mitochondrial proteins
In the respiratory chain of rat skeletal muscle, most carbony-
lated subunits were found in complexes I and III [81], the sites
of the major part of mitochondrial ROS production [37].
However, carbonylation has also been found in subunits of
the other respiratory complexes [52,81,87–89], which produce
only small amounts of ROS (complexes II and IV) or none at all
(complex V) [37]. Likewise many matrix enzymes are carbo-
nylated although they are not themselves ROS producers
[52,81,86,88]. As discussed in Section 2, it requires an
“unprotected” metal ion as well as H2O2 to give the Fenton
reaction and, since superoxide and H2O2 diffuse throughout
the inner membrane and matrix, the carbonylated proteins
may indicate which proteins are neighbours when metal ions
are released? A major site of iron ion release appears to
0
5
10
15
20
25
Distance (amino acid)
% o
f s i t e s
Actual
Simulated
1 10 100 1 10 100
Distance (amino acid)
Actual
Simulated
Simulated surface exposed
Simulated RKPT rich
A B
Fig. 1 – Monte Carlo simulation of distance between carbonylation sites. A. Over all, simulated profile of distance between
potential carbonylation RKPT (Arg, Lys, Pro, Thr) sites is nearly identical to the actual pattern observed. B. The proportion of
carbonylated sites at lower distance (below 10 amino acids) is very low as per random mechanism. The proportion is slightly
higher when carbonylated sites are considered to be in highly surface exposed regions. However, the simulation pattern is
quite similar to the actual one when carbonylated sites are assumed to be in RKPT rich regions. Monte Carlo simulations were
performed programmatically in Python scripting language (ver. 2.6). Pseudo-random numbers were generated to match the
actual number of RKPT sites or carbonylated sites in a given protein. For simulating RKPT sites, numbers were drawn from
within the range of protein length whereas for simulating carbonylation sites they were drawn from the list of actual positions
of RKPT sites in proteins. Sites (centered in 21-residue window) with hydropathy score (using Kyte-Doolittle scale) below −0.5
were assumedto be in highly surface exposed regions. RKPT rich sites were considered as sites withat least four RKPT residues
within a moving window of seven residues around the RKPT site. Simulations were run for 100,000 iterations.
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be aconitase, which is very sensitive to superoxide (see
Section 2.5). If the Krebs cycle is organized in a metabolon in
the matrix [90], then one would expect many Krebs cycle
enzymes to be carbonylated under oxidative stress because of
the proximity of aconitase. Indeed many Krebs cycle enzymes
are known to be carbonylated [52,81,86,88]; however, these
enzymes are quite abundant and would also be expected to be
oxidized frequently if a random diffusion-collision model wasapplied. Although we know that oxidative stress generally
leads to loss of enzymatic function e.g., [86,91], in most cases
we do not know how carbonylation at specific sites affects the
function of the modified protein.
It has been suggested that Fe ions replace Mg 2+ at binding
sites on the carbonylated proteins or on interacting nucleo-
tides and that the MCO damage occurs near these sites [86,92].
An Fe ion binding instead of Mg 2+ could well be “unprotected”
and able to catalyze the Fenton reaction, since the coordina-
tion requirements of the two metals differ markedly.
In two studies, N-formylkynurenine – doubly oxidized Trp
residues – sites were identified in mitochondrial proteins and
taken as an indicator of in vivo protein oxidation [57,58].
Although it hasrecently been shownthat Trp oxidation can be
a preparation artifact occurring during gel electrophoresis [61],
that may not be true for all oxidized Trp. We also need to keep
in mind that DNPH may react with both singly and doubly
oxidized Trp residues. In fact, nine of the ten rice mitochon-
drial proteins containing N-formylkynurenine had previously
been identified as carbonylated proteins although the site of
carbonylation was not identified [52,58]. Overall, the pattern of
proteins containing oxidized Trp was similar to that of
carbonylation with modification of many subunits of electron
transport complexes and Krebs cycle enzymes.
5.2. Carbonylation via conjugation with oxidized
carbohydrates and fatty acids
Another mode of protein carbonylation is by formation of AGE
adducts. In the presence of high levels of reducing carbohy-
drates, peptides and proteins can become glycated by forming
a Schiff base, which further rearranges to stable Amadori
products that contain carbonyl residues [9]. Oxidative degra-
dation of these products can lead to extensive formation of
carbonyls, which induce protein cross-linking and aggregation
in the cell. Formation and accumulation of AGE adducts is a
major route of protein carbonylation in untreated diabetes
[93], but it is also known to be associated with ageing and
chronic diseases such as Alzheimer's disease [94] and vascular
dementia [95]. Table 2b lists the main AGE products.
Protein carbonylation, primarily on Lys residues, can also
be generated by secondary reaction with aldehydes produced
during PUFA peroxidation. Some of these ALE products are
listed in Table 2b. Cells contain a number of carbonyl
reductases, distributed in all compartments including the
mitochondria, which help to remove low-molecular-weight
compounds containing reactive carbonyl groups before they
can form conjugates with proteins and other macromolecules
[96]. A mitochondrial aldehyde dehydrogenase restores
pollen fertility in cytoplasmic male sterile maize (CMS-T)
possibly because it removes the aldehydes formed by PUFA
oxidation before they can form conjugates with mitochon-
drial proteins and in that way prevent oxidative damage
[97,98]. HNE-conjugation to a number of mitochondrial pro-
teins including subunits of mitochondrial electron transport
complexes has been reported [87,99]. When tested on
mitochondrial enzymes, HNE-conjugation had little or no
effect on the activity of the affected enzyme, but the
association between electron transport complexes appeared
to be affected [99].It is becoming evident that there exists a close interplay
between the different types of protein carbonylation and MCO,
but the physiological mechanisms controlling these processes
are not yet completely understood. It has been shown that
MCO and free radicals play a major role in the formation of
AGEs and AGE-induced protein cross-linking [7,8]. AndMCO of
PUFA in the presence of proteins can also lead to N(6)-
carboxymethyllysine (CML) formation suggesting that MCO
plays a role in ALE formation as well [9]. In senescent human
fibroblast a number of oxidatively modified proteins –
carbonylated, HNE-conjugated, AGE-modified – were detected;
half of them were of mitochondrial origin [100]. Several
structural proteins (e.g. vimentin) were found to contain
both AGE and HNE modifications.
5.3. Protein turnover
Whenever quantifying the amount of a component in a
biological system and its changes with time, we need to
keep in mind that we are observing steady-state levels, which
are the net result of synthesis and degradation (e.g., [101])
Mitochondrial protein carbonylation is a case in point, since
we know that damaged mitochondrial proteins are degraded
by specific proteases [102]. It is therefore entirely possible that
proteins with a low steady-state level of carbonylation in fact
have a very high turnover rate. We do not have any
information about that at the moment.
The quantification of protein carbonylation using the
methods described above typically give values of 1–4 nmol/
mg of protein rising to 8 nmol/mg of protein under oxidative
stress e.g. caused by disease or age (e.g., [69,103] and
references therein). This is equivalent to 0.05–0.4 carbonyl
per 50 kDaprotein or, expressed differently, as many as 40% of
all protein molecules have one carbonyl group. This is a high
value and the cost of replacing the proteins must be heavy
burden for cells. In mitochondria, which are one of the centres
of oxidative stress, protein turnover may cost as much as 2–
20% of the total energy output [103].
We know very little about the extent to which MCO
contributes to protein turnover. The turnover times of three
mitochondrial proteins, uncoupling protein, Mn-SOD and
peroxiredoxin IIf were reported to be 6 h, 72 h and 72 h,
respectively [104]. They differ markedly in their metal binding
and ROS interaction. The uncoupling protein does not contain
bound metal ion and it is activated by superoxide via an
indirect process [105,106]. Mn-SOD containing an Mn ion
converts superoxide to H2O2, while peroxiredoxin IIf contain-
ing no bound metal ion reduces a wide range of peroxides
using a pair of Cys residues [107]. Mn-SOD and peroxiredoxin
were both reported to be carbonylated in potato tuber
mitochondria [52], which could be due to the conjugation
with HNE originating from superoxide-mediated PUFA
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oxidation [106]. In apple fruit mitochondria only the MnSOD
was observed to be carbonylated [88], in rat skeletal muscle
mitochondria only peroxiredoxin was observed to be carbo-
nylated [81] while Mn-SOD contained an oxidized Trp residue
in rice leaf mitochondria [58] but not in human or bovine heart
mitochondria [57]. These few and disjointed observations do
not give a clear picture of the contribution of MCO to protein
turnover and more focussed experiments are required.
5.4. Intracellular signalling
ROS not only cause damage to cellular components, but they
are also involved in intracellular signalling and H2O2 is often
presented as being the most likely messenger (e.g., [108])
partly because it can cross membranes through aquaporins
[85]. However, H2O2 does not have the requisite information
content to allow the interaction partner to identify its point of
origin. It has therefore been proposed that irreversibly
(carbonylated) peptides deriving from the proteolytic degra-
dation of oxidized proteins can be the specific secondary ROS
messengers from organelles such as mitochondria or chloro-
plasts [109]. In this connection it is interesting that carbonyl-
ation and subsequent proteolytic degradation of annexin A1
has been suggested to be involved in endothelin-mediated cell
growth and survival although the mechanism by which the
signal is transmitted is unknown [110,111].
6. Summary
MCO is a common cause of irreversible protein modification,
which increases in living cells as a result of stress, age and
disease. It occurs when a metal ion, often Fe3+ or Cu2+, is
released from its normal protected environment and binds to
an unprotected site. There it interacts with H2O2 in the Fenton
reaction to produce hydroxyl radicals, which oxidize adjacent
aminoacid side chains. Carbonyl groupsare formed on several
amino acids and this modification can be tagged, e.g. by
reaction with hydrazide derivatives, which in turn can be
recognized e.g. by use of antibodies. This recognition can be
used for quantification of protein carbonylation and in
proteomic strategies (with or without 2-DE) where the
carbonylated proteins are enriched, separated and identified
by use of LC-MS/MS methods. The large number of possible
oxidative modifications makes it particularly difficult to
identify peptides with specific modifications. In spite of this
about 450 carbonylation sites have been identified and they
have a tendency to cluster in RKPT-rich regions. The
mitochondrion is a major site of ROS production in the cell
and many carbonylated mitochondrial proteins have been
identified. Some of these are close to the sites of ROS
production, but many are not and may instead be close to
sites of metal ion release. The carbonylated mitochondrial
proteins are degraded by dedicated proteases, but the extent
to which this contributes to mitochondrial protein turnover is
not understood. We also know very little about the effect of
carbonylation on the properties of theaffected proteins and on
their turnover.
Supplementary materials related to this article can be
found online at doi:10.1016/j.jprot.2011.05.004.
Acknowledgements
We are grateful to Dr. Pai Pedas for making unpublished data
available, to Morten J. Bjerrum and Pai Pedas for useful
suggestions and to Dr. Kristian Kristensen for help with the
statistical analysis. This work was supported by a grant from
the Faculty of Agricultural Sciences, Aarhus University to IMMand by European 6th Framework Program Grant Proteomage
contract no. LSHMCT-518230 to ARW.
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