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

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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].

2229 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


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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


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


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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Protein Carbonylation and Metal-catalyzed Protein Oxidation in a Cellular Perspective