Journal of Chromatography B, 1019 (2016) 178–190
Contents lists available at ScienceDirect
Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb
A step-by-step protocol for assaying protein carbonylation in
biological samples
Graziano Colombo a,∗ , Marco Clerici a , Maria Elisa Garavaglia a , Daniela Giustarini b ,
Ranieri Rossi b , Aldo Milzani a , Isabella Dalle-Donne a
a
b
Department of Biosciences, Università degli Studi di Milano, Milan, Italy
Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Siena, Italy
a r t i c l e
i n f o
Article history:
Received 12 October 2015
Received in revised form
24 November 2015
Accepted 28 November 2015
Available online 2 December 2015
Keywords:
Protein carbonylation
2,4-Dinitrophenylhydrazine
Biotin-hydrazide
Aldehyde-reactive probe
Fluorescein-5-thiosemicarbazide
a b s t r a c t
Protein carbonylation represents the most frequent and usually irreversible oxidative modification affecting proteins. This modification is chemically stable and this feature is particularly important for storage
and detection of carbonylated proteins. Many biochemical and analytical methods have been developed during the last thirty years to assay protein carbonylation. The most successful method consists
on protein carbonyl (PCO) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and consequent spectrophotometric assay. This assay allows a global quantification of PCO content due to the ability of DNPH
to react with carbonyl giving rise to an adduct able to absorb at 366 nm. Similar approaches were also
developed employing chromatographic separation, in particular HPLC, and parallel detection of absorbing adducts. Subsequently, immunological techniques, such as Western immunoblot or ELISA, have been
developed leading to an increase of sensitivity in protein carbonylation detection. Currently, they are
widely employed to evaluate change in total protein carbonylation and eventually to highlight the specific
proteins undergoing selective oxidation. In the last decade, many mass spectrometry (MS) approaches
have been developed for the identification of the carbonylated proteins and the relative amino acid
residues modified to carbonyl derivatives. Although these MS methods are much more focused and
detailed due to their ability to identify the amino acid residues undergoing carbonylation, they still
require too expensive equipments and, therefore, are limited in distribution. In this protocol paper, we
summarise and comment on the most diffuse protocols that a standard laboratory can employ to assess
protein carbonylation; in particular, we describe step-by-step the different protocols, adding suggestions
coming from our on-bench experience.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations:
2D-GE, two-dimensional gel electrophoresis; 4-HNE, 4hydroxynonenal; ARP, aldehyde-reactive probe; BHZ, biotin-hydrazide; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DMF, dimethyl
formammide; DMSO, dimethyl sulfoxide; DNPH, 2,4-dinitrophenylhydrazine;
DTT, dithiotreitol; ECL, enhanced chemiluminescence; ELISA, enzyme-linked
immunosorbent assay; FITC, fluorescein isothiocyanate; FTC, fluorescein-5thiosemicarbazide; HGFs, human gingival fibroblasts; HPLC, high performance
liquid chromatography; HRP, horseradish peroxidase; HSA, human serum albumin;
IAM, iodoacetamide; IEF, isoelectric focusing; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MS, mass spectrometry; MW, molecular weight;
PBST, phosphate buffered saline with Tween-20; PCO, protein carbonyls; PNGase
F, peptide N-glycosidase F; PVDF, polyvinylidene fluoride; ROS, reactive oxygen
species; RT, room temperature; SDS, sodium dodecyl sulphate; SDS-PAGE, sodium
dodecyl sulphate polyacrylamide gel electrophoresis; TBP, tri-butyl phosphine; TCA,
trichloroacetic acid; UTC, urea-thiourea-CHAPS solution.
∗ Corresponding author at: Department of Biosciences, University of Milan, via
Celoria 26, I-20133 Milan, Italy. Fax: +39 2 50314781.
E-mail address: graziano.colombo@unimi.it (G. Colombo).
http://dx.doi.org/10.1016/j.jchromb.2015.11.052
1570-0232/© 2015 Elsevier B.V. All rights reserved.
The introduction of reactive carbonyl groups (CO), mainly aldehydes and ketones, into a protein structure is defined “protein
carbonylation” [1]. Protein carbonylation includes many chemical modifications occurring through different reaction mechanisms
that can be summarised as follows:
1) Direct oxidation of several amino acid residues [2,3] induced by
hydroxyl radical (HO• ): this radical can be generated by Fenton reaction of metal cations with hydrogen peroxide [1,4] or
by ionizing radiations [5]. HO• induces oxidation of proline,
arginine, lysine, and threonine residue side chains to aldehydes or ketones. Two typical products of oxidation are glutamic
semialdehyde and aminoadipic semialdehyde from arginine and
lysine oxidation, respectively [6].
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
2) Protein backbone hydrolysis: hydroxyl radical attack to protein
backbone can induce hydrolysis through ␣-amidation pathway
[7].
3) “Michael addition” reactions: cysteine, histidine, and
lysine residues can react with carbonyl species, such as
4-hydroxynonenal (4-HNE), 2-propenal (acrolein), and
malondialdehyde, generated during lipid peroxidation [8].
4) Glycation/glycoxidation reactions: the amino group of lysine
residues can react with reducing sugars or their oxidative products to generate carbonyl species such as carboxymethyl lysine
[9].
Detection and quantification of protein carbonyls (PCO) in biological samples is an indirect way to determine the level of oxidative
stress. At the current time, protein carbonylation is a widely occurring and accepted irreversible marker of protein oxidation and
hundreds of published papers reported PCO quantification.
There are many methods used nowadays for evaluation of
the content of carbonylated proteins and, among them, the most
employed is based on 2,4-dinitrophenylhydrazine (DNPH) and was
originally developed by Levine et al. [10]. This molecule reacts
with carbonyl groups leading to the formation of the stable 2,4dinitrophenylhydrazone. The dinitrophenyl group (DNP) can be
detected and quantified spectrophotometrically because it is characterized by a typical absorption spectrum with a maximum at
365–375 nm [10]. In addition, spectrophotometric measurement
of DNP can also be used in association with HPLC protein separation adding the possibility to detect more precisely the proteins
undergoing carbonylation [11,12].
Within a few years from the fundamental paper of Levine et al.
[10], the development of good antibodies able to recognize the
DNP adducts has opened the possibility to increase the sensitivity
of PCO detection. The only use of the high recognition specificity
of anti DNP antibodies or the combination with other standard
protein separation and/or detection methods (e.g. electrophoretic
techniques) allowed researchers to detect protein carbonylation
with dot blot, immunochemistry, ELISA and Western blot protocols. Today, hundreds of papers investigating PCO and using these
well ascertained methods are reported in literature [13–17].
A more recent and powerful approach consists on using MS techniques as investigation tools. These methods are not accessible
to all laboratories due to the high cost for analysis instrumentation. Although these methods are very sensitive and can identify
specific oxidized residues without, in theory, requiring protein
labelling, in practice, protein labelling is often used in mass spectrometric analysis because it has the advantage to allow protein
enrichment (e.g. biotin-hydrazide derivatization of proteins can
be followed by affinity chromatography column enrichment with
immobilized streptavidin). For instance, using a biotin-hydrazide
based approach, it was possible to identify 100 carbonylated proteins, including low abundance receptors, in brain homogenates
179
of mice of different ages [18]. Alternatively, label-free approaches
have also been developed as reported by capture of carbonylated
peptides by a solid-phase hydrazide reagent [19] or by immobilized
oxalyldihydrazide on a microchip [20].
Carbonyl content of purified proteins is usually expressed
as moles carbonyl/mole protein. Otherwise, for cell or tissue
homogenates, protein carbonyl content is expressed as nmol carbonyl/mg protein. This means that both carbonyl levels and protein
levels need to be accurately determined. Carbonyl content can
be indirectly measured evaluating DNPH incorporation using a
spectrophotometer (Section 2.2), while protein amount is usually
determined by protein assay.
In case of immunowestern blot analysis (Sections 2.4 and 2.5),
the carbonyl levels can be densitometrically defined using specific
software, whereas the protein levels are usually evaluated after
Amido Black or Coomassie blue stain of western blot membrane.
The ratio between the carbonyl signal intensity and the protein
signal intensity will give specific carbonyl content. In general, in
this kind of experiment, many researchers consider sufficient to
express protein carbonyl content in treated samples as a percentage
increase/decrease respect to control one. In case a more accurate
quantification is required and protein carbonyl content needs to be
expressed as nmol carbonyl/mg protein, an oxidized protein standard is required to compare sample signals to well known carbonyl
protein content standard.
The protein carbonyl content highly increases under pathological conditions related to oxidative stress. For example, in
plasma proteins of children with different forms of juvenile
chronic arthritis, the carbonyl content was significantly higher
than in healthy group (1.36 ± 0.68 vs. 0.807 ± 0.16 nmol carbonyl/mg of protein) [21]. A more evident difference was
observed comparing plasma protein carbonyl groups among normal volunteers (0.76 ± 0.51 ␮mol/l), patients with chronic renal
failure (13.73 ± 4.45 ␮mol/l) and patients on chronic maintenance
haemodialysis (16.95 ± 2.62 ␮mol/l) [22].
In this article, we describe in a user-friendly and step-bystep way, with detailed guidelines coming from our on-bench
experience, the most used methods to detect and quantify protein carbonylation in a standard research laboratory devoted to
studying protein oxidative modifications and whose equipment
is composed by relatively cheap and highly diffuse instrumentation (e.g., spectrophotometer, SDS-PAGE, 2D-GE and Western blot
apparatuses).
2. Protocols
2.1. PCO labelling with DNPH
The molecule DNPH, also known as Brady’s reagent, is a specific probe able to react with PCO leading to the formation of
protein-conjugated dinitrophenylhydrazones (DNP) (Fig. 1). These
Fig. 1. DNPH reaction with carbonyl species. DNPH reacts readily with aldehydes and ketones via a condensation reaction to produce the corresponding hydrazine. Modified
from [11]. Copyright© 2013 Elsevier Ireland Ltd.
180
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
protein-DNP adducts are characterized by a peak absorbance at
366 nm, which makes DNPH employment easy and useful in order
to perform a quantitative determination of PCO content by spectrophotometer. In addition, DNP adducts are recognized by specific
primary antibodies, thus allowing the use of DNPH to derivatize
PCO in association with many electrophoresis techniques and western blot methods.
2.2. Spectrophotometric detection of PCO using DNPH
2.2.1. Required chemicals
• 10 mM DNPH solution in 2 N HCl (see Supplementary)
• Protein sample solution
(Advice: According to our experience, we suggest to start the
protocol with a protein sample concentration of 1 mg/ml because,
after derivatization with DNPH and protein precipitation, higher
protein concentrations could lead to formation of pellets very difficult to resuspend).
Note: Prepare fresh DNPH solution each time because reactivity could decrease over time [44]. Keep the DNPH solution at RT
protected from light because DNPH is light-sensitive.
• 2 N HCl (see Supplementary)
• 20% (v/v) trichloroacetic acid (TCA) solution, ice-cold (see Supplementary)
• 1:1 (v/v) ethanol:ethyl acetate
• 6 M guanidine hydrochloride (see Supplementary) or, in alternative,
• 0.2% (w/v) SDS in 20 mM Tris–HCl, pH 6.8 (see Supplementary)
• bicinchoninic acid (BCA) protein assay kit
2.2.2. Procedure
1. Add 200 ␮l of 10 mM DNPH solution to 1-ml protein samples.
2. Prepare a blank sample adding 200 ␮l of 2 N HCl (without
DNPH) to 1 ml of protein sample.
3. Vortex-mix samples and leave them in the dark at room temperature (RT) for 60 min, with vortex-mixing every 10–15 min.
4. Add a volume (1.2 ml) of 20% TCA solution to protein samples
and incubate on ice for 15 min.
5. Centrifuge samples at 10,000 × g in a tabletop microcentrifuge
for 5 min, at 4 ◦ C.
6. Discard supernatants, wash protein pellets once with 1 ml of
20% TCA and vortex-mix.
7. Collect pellets centrifuging at 10,000 × g for 5 min, at 4 ◦ C.
8. Discard supernatants, wash protein pellets with 1 ml of 1:1
(v/v) ethanol:ethyl acetate and mix by vortexing in order to
remove any free DNPH.
9. Repeat Steps 6 and 7 at least twice until supernatants are completely transparent.
(Advice: Extend washes to completely remove any free DNPH,
otherwise residual DNPH in the protein pellet could lead to
overestimation of sample PCO content).
10. Collect pellets centrifuging at 10,000 × g for 5 min, at 4 ◦ C and
discard supernatants.
11. Let the pellets vacuum dry for about 5 min to allow complete
solvent evaporation.
(Advice: Constantly check pellets during vacuum drying to
avoid overdrying, which could make pellet resuspension harder
to obtain and incomplete).
12. Resuspend protein pellets in 1 ml of 6 M guanidine hydrochloride (dissolved in 50 mM phosphate buffer, pH 2.3) incubating
at 37 ◦ C for 15–30 min with vortex mixing.
Note: Protein pellets can alternatively be resuspended in
20 mM Tris–HCl, pH 6.8, containing 0.2% (w/v) SDS in case of
protein quantification with the BCA protein assay and subsequent SDS-PAGE (see Section 2.3: Direct in-solution PCO
derivatization with DNPH).
13. Once protein pellets are completely dissolved, carbonyl contents can be determined from the peak absorbance at ∼366 nm
by using a molar absorption coefficient of 22,000 M−1 cm−1 . The
protein sample incubated with 2 N HCl without DNPH should be
used as a blank in order to subtract intrinsic protein absorbance
to the absorbance of specific DNP-adducts.
(Advice: Scan samples in the wavelength range from 300 nm
to 500 nm by a spectrophotometer in order to singularly check
the profile quality of all sample spectra and choose the correct
maximum peak).
14. To calculate PCO concentration in cuvette (expressed as ␮M) or
sample carbonyl content (expressed as nmol PCO/mg protein),
use the following formulas:
PCO concentration(CUVETTE) = [PCO](M)
= 106 ×
PCO concentration(SAMPLE)
[PCO]
=
nmol
mgprotein
= 106 ×
Abs366nm
22, 000M−1 cm−1
Abs366nm
22,000M−1∗ cm−1
[protein]mg/ml
where:Abs366 nm = sample
absorbance
at
coefficient
(␧)
at
366 nm22,000 M−1 cm−1 = extinction
366 nm[protein] (mg/ml) = protein concentration expressed in
mg/ml
2.2.3. DNPH alkaline method
Recently, an alternative to the reference DNPH spectrophotometric assay was proposed [23]. In this modified DNPH
spectrophotometric assay, NaOH is added to the protein solution
after the addition of DNPH, thus causing the shift of the maximum absorbance wavelength of the derivatized proteins from
∼370 to 450 nm. This shift in the absorbance maximum minimizes
the interference of free DNPH, which absorbs at 366–370 nm as
DNP derivatives of carbonyl groups, and allows the direct quantification of PCO in the sample solution avoiding protein precipitation,
washing, and resuspension steps.
Caution: Considering the instability of DNP in alkaline medium,
the incubation time (10 min) after NaOH addition must be rigorously controlled [23].
Note: nucleic acid contamination. Nucleic acid contamination
causes serious artifactual increase in the PCO measurement
determined by spectrophotometric techniques. Both in vitro synthesized DNA oligonucleotides and purified chromosomal DNA
react strongly with DNPH. Treatment of cell extracts with DNase
and RNase or with streptomycin sulphate to precipitate nucleic
acids markedly reduces the measured carbonyl content [24]. Alternatively, mild protein extraction protocols, e.g., using hypotonic
lysis buffers and avoiding strong detergents and sonication, may
be employed to reduce disruption of nuclei or mitochondria and
leakage of nucleic acids [24].
Note: carbohydrate contamination. Reduced carbohydrates may
also contain carbonyl groups that can potentially interfere with the
protein carbonylation measurements. It is possible to selectively
remove carbohydrates by protein extracts, e.g., by lectin affinity
or using protein specific extraction methods like TCA precipitation
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
following deglycosylation by peptide N-glycosidase F (PNGase F)
[25].
Note: cross-reaction of DNPH. DNPH can also react with sulphenic
acids [26]. Sample pre-treatment with a mild reductant such as tributyl phosphine (TBP), a molecule able to reduce mildly oxidized
thiols, will reduce the contribution of the thio-aldehydes to the
DNPH assay results [25].
2.3. Direct in-solution derivatization of proteins with DNPH
2.3.1. Required chemicals
• 10 mM DNPH solution in 2 N HCl (see Supplementary)
• Protein sample solution
(Advice: According to our experience, start the protocol with
a protein sample concentration of 1 mg/ml because, after DNPH
derivatization and protein precipitation, higher protein concentrations could lead to formation of protein pellets very difficult to
resuspend).
Note: Prepare fresh DNPH solution each time because reactivity could decrease over time [44]. Keep the DNPH solution at RT
protected from light because DNPH is light-sensitive.
•
•
•
•
•
2 N HCl (see Supplementary)
20% (v/v) TCA solution, ice-cold (see Supplementary)
1:1 (v/v) ethanol:ethyl acetate (EtOH:EtAc)
0.2% (w/v) SDS in 20 mM Tris–HCl, pH 6.8 (see Supplementary)
BCA protein assay kit
2.3.2. Procedure
1. Add 200 ␮l of 10 mM DNPH solution to 1 ml of 1 mg/ml protein
samples.
2. Vortex-mix samples and leave them in the dark at RT for 60 min,
with vortex-mixing every 10–15 min.
3. Add a volume (1.2 ml) of 20% TCA solution to protein samples
and incubate on ice for 15 min.
4. Centrifuge samples at 10,000 × g in a tabletop microcentrifuge
for 5 min, at 4 ◦ C.
5. Discard supernatants, wash protein pellets once with 1 ml of
20% TCA and vortex-mix.
6. Collect pellets centrifuging at 10,000 × g for 5 min, at 4 ◦ C.
7. Discard supernatants, wash protein pellets with 1-ml aliquots
of 1:1 (v/v) ethanol:ethyl acetate and mix by vortexing in order
to remove any free DNPH.
8. Repeat steps 5 and 6 at least twice.
9. Collect pellets centrifuging at 10,000 × g for 5 min, at 4 ◦ C and
discard supernatants.
10. Let the pellets vacuum dry for about 5 min to allow complete
solvent evaporation.
(Advice: Constantly check pellets during vacuum drying
because overdrying could make pellet resuspension harder to
obtain).
11. Resuspend protein pellets in 1 ml of 0.2% (w/v) SDS in 20 mM
Tris–HCl, pH 6.8, incubating at 95 ◦ C for 5–10 min to complete
resuspension.
Note: Protein pellets can alternatively be resuspended in 1×
Laemmli sample buffer (see Supplementary) in case protein
quantification with the BCA assay is not required.
12. Once protein pellets are completely dissolved, protein concentration can be measured using the BCA protein assay;
subsequently, electrophoretic separation can be performed.
181
2.4. Immunoblot detection of PCO after SDS-PAGE separation
After derivatization with DNPH (see Section 2.3), carbonylated
proteins can be separated by SDS-PAGE according to standard techniques. Once the proteins are electrotransferred from the gel to a
PVDF membrane, it is possible to proceed with a two-steps immunodetection protocol employing primary anti-DNP antibodies (e.g.,
anti-dinitrophenyl-KLH antibodies, rabbit IgG fraction (cod. A6430)
from Molecular Probes (Eugene, OR, USA)) and horseradish peroxidase (HRP)-conjugated secondary antibodies (e.g., goat anti-rabbit
IgG, (cod. G21234) from Molecular Probes).
2.4.1. Required chemicals and equipment
• DNPH-derivatized proteins (see Section 2.3)
• Minigel electrophoresis unit and transfer unit (e.g., from Bio-Rad)
(see Supplementary for gel formulation and transfer buffer composition)
• PBS (pH 7.2) with 0.1% Tween-20 (PBST) (see Supplementary)
• 5% (w/v) non-fat dry milk in PBST
• Primary antibody (e.g., rabbit anti-DNP antibody)
• HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG)
• ECL Western blotting detection reagents (e.g., from GE Healthcare)
• Immobilon-P membranes (e.g., from Biorad)
• Plastic wrap, 100 ␮m thick
• X-ray film
2.4.2. Procedure
1. Dilute protein sample in 2× Laemmli sample buffer (see Supplementary) and heat samples at ∼95 ◦ C for 5 min.
2. Separate 10–20 ␮g of DNPH-labelled proteins by SDS-PAGE
in a minigel electrophoresis unit according to manufacturer’s
instructions.
3. Transfer the electrophoretically separated proteins from the
SDS-PAGE gel onto a PVDF membrane by electroblotting using
a transfer unit according to manufacturer’s instructions.
Caution: Transfer buffer contains methanol, a very volatile
and toxic solvent. It should be handled in a fume hood and
all the steps necessary to transfer proteins to PVDF membrane
should be performed in a fume hood.
(Advice: In our hands, performing tank transfer rather than
semidry transfer is usually associated with a better transfer
efficiency).
(Advice: After SDS-PAGE separation, wash gels in transfer
buffer (see Supplementary) for 15 min in order to eliminate SDS
residues and obtain a clearer background during ECL development).
4. After protein transfer to a PVDF membrane, wash the membrane for 15 min in PBST and continue with immunodecoration.
(Advice: In case the PVDF membrane should be stored for a
long period of time, after transfer to PVDF, wash the membrane
for 5 min in methanol and let it air dry. Store the PVDF membrane at −20 ◦ C until use. To restart the immunodecoration
protocol, equilibrate the PVDF membrane in PBST for at least
60 min in order to allow a complete rehydration and continue
with step 5).
5. Block non-specific binding sites on the membrane by equilibrating it in 5% (w/v) non-fat dry milk in PBST for at least
60 min.
6. Wash the membrane thrice with abundant PBST for 10 min
each.
7. Incubate the membrane in primary antibody solution for 2 h, at
RT, with gentle agitation.
Note: Primary antibody solution is prepared by diluting
the primary antibody in 5% (w/v) non-fat dry milk in PBST.
182
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
Fig. 2. Indirect on-membrane protein labelling with DNPH of purified human serum albumin (HSA). Total carbonyl formation in cigarette smoke extract (CSE)-treated HSA.
HSA solutions were treated with vehicle (control) or 1%, 4%, 16%, and 64% (v/v) CSE and exhaustively dialyzed against PBS. (A) The increase in HSA carbonyl content was
assessed as DNP-protein adducts with Western immunoblotting using anti-DNP antibodies and subsequently visualized with ECL. (B) The same PVDF membrane stained
with Amido Black after ECL development. (C) Densitometric analysis of carbonyl content. Changes in carbonylation signal in comparison with carbonylation of control HSA,
considered as 100%, are shown. Reprinted with permission from [26]. Copyright© 2013 Elsevier Ireland Ltd.
8.
9.
10.
11.
12.
13.
14.
15.
Antibody dilution is highly variable; for instance, we use
anti-dinitrophenyl-KLH antibodies, rabbit IgG fraction (cod.
A6430) from Molecular Probes at 1:40,000 dilution (in combination with secondary goat anti-rabbit IgG, HRP conjugate
(cod. G21234) from Molecular Probes), which is sufficient to
develop a signal in ECL within 10 min. If a particularly weak
signal is expected, overnight primary antibody incubation, at
4 ◦ C, with gentle agitation, is the better choice to improve ECL
signal.
Note: Use antibody solution once and prepare it just before
use.
Wash the membrane thrice with abundant PBST for 10 min
each.
Incubate the membrane with secondary antibody at RT for at
least 1 h. We use secondary goat anti-rabbit IgG, HRP conjugate (cod. G21234) from Molecular Probes at 1:80,000 dilution,
which is sufficient to develop a signal in ECL within 10 min (in
combination with primary rabbit IgG fraction (cod. A6430) from
Molecular Probes).
Note: Use antibody solution once and prepare it just before
use.
Wash the membrane thrice with abundant PBST for 10 min
each.
Cover the membrane with ECL working solution, according to
ECL manufacturer’s instruction. In general, depending on the
ECL kit, 1–5 min is enough time for the reaction to develop.
Discard excess of solution, wipe the back of the membrane with
a paper towel and wrap the membrane between two plastic
sheets, 100 ␮m thick.
Be sure that no air bubbles are trapped between the membrane and plastic wrap on the side to be exposed to X-ray film;
proceed immediately to expose the membrane to X-ray film.
Positive and negative controls are highly recommended in
order to check, respectively, proper reactivity of anti-DNP antibodies and to exclude any non-specific reactivity not due to
DNPH binding to PCO.
After ECL development, disassemble the plastic sandwich and
rinse abundantly the membrane in PBST in order to wash away
all ECL reagents.
Fig. 3. Indirect on-membrane labelling with DNPH of proteins from cultured cell
extracts. Total PCO formation in CSE-treated human gingival fibroblasts (HGFs). Cells
were treated without (control) or with 0.5-2-5-12 puffs of cigarette smoke. MW represents protein molecular weights. The five lanes named “anti PCO” represent the
signal after ECL development and show the increase in protein carbonylation in parallel with the increasing of cigarette smoke dose. The five lanes named “Amido Black”
represent the staining of the same PVDF membrane after washing and staining with
Amido Black according to the described protocol. Modified from [15]. Copyright©
2013 Elsevier Ireland Ltd.
16.
17.
18.
19.
(Advice: We suggest washing the membrane for at least 1 h
with frequent changes of PBST).
Proceed to stain the membrane with Amido Black (see Supplementary for Amido Black stain solution and Amido Black destain
solution).
Caution: Methanol is toxic and acetic acid is corrosive; these
solvents should be handled in a fume hood. Preparation of
staining and destaining solutions for Amido Black should be
performed in a fume hood and also all the steps for staining
(from 16 to 20) should be performed in a fume hood.
Incubate the membrane for 5 min in Amido Black stain solution.
Transfer the membrane in Amido Black destain solution and
rinse for 5 min.
Change the Amido Black destain solution every 5 min and proceed until the membrane background is white (Fig. 3).
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
20. Lay the membrane on a paper towel under a fume hood and let
it air dry.
7.
2.5. Indirect on-membrane protein labelling with DNPH
Derivatization of carbonylated proteins with DNPH is also
possible after protein separation with electrophoretic standard
techniques and transfer to PVDF membrane. Once the proteins are
electro-transferred from the gel to a PVDF membrane, an easy onmembrane derivatization can be performed (Figs. 2 and 3).
2.5.1. Required chemicals and equipment
• 0.1 mg/ml DNPH solution in 2 N HCl (see Supplementary)
• PBS (pH 7.2) with 0.1% Tween-20 (PBST) (see Supplementary)
• 5% (w/v) non-fat dry milk in PBST
• Primary antibody (e.g., rabbit anti-DNP antibody)
• HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG)
• ECL Western blotting detection reagents (e.g., from GE Healthcare)
• Plastic wrap, 100 ␮m thick
• X-ray film
Note: Prepare fresh DNPH solution each time because reactivity could decrease over time [44]. Keep the DNPH solution at RT
protected from light because DNPH is light-sensitive.
8.
9.
10.
11.
12.
2.5.2. Procedure
1. Transfer electrophoresed proteins from the SDS-PAGE gel onto
a PVDF membrane by electroblotting using a transfer unit
according to manufacturer’s instructions.
Caution: Transfer buffer contains methanol, a very volatile
and toxic solvent. It should be handled in a fume hood and
all the required steps to transfer proteins to PVDF should be
performed in a fume hood.
(Advice: In our hands, performing tank transfer rather than
semidry transfer is usually associated with a better transfer
efficiency).
(Advice: In our lab, after SDS-PAGE separation, we usually
wash gels in transfer buffer (see Supplementary) for 15 min in
order to eliminate SDS residues and obtain a clearer background
during ECL development).
2. After transfer, wash the membrane for 15 min in PBST.
(Advice: In case the PVDF membrane must be stored for long
periods of time, after transfer, wash the membrane for 5 min in
methanol, let it air dry and store it at −20 ◦ C until use. To restart
the immunodecoration protocol, equilibrate the PVDF membrane in PBST for at least 60 min in order to allow a complete
rehydration and continue with step 3).
3. Incubate the PVDF membrane in 2 N HCl for 5 min in order to
equilibrate it for successive derivatization steps.
Caution: HCl is harmful and highly corrosive; use all the
required personal protective equipment such as latex gloves,
protective eye goggles, chemical-resistant clothing and shoes
to avoid irreversible damages to respiratory organs, eyes, skin,
and intestines.
4. Discard 2 N HCl and incubate the PVDF membrane in 0.1 mg/ml
DNPH solution in 2 N HCl for 5 min to derivatize PCO.
5. Discard DNPH solution and wash thrice the PVDF membrane in
2 N HCl for 5 min each.
6. Wash seven times the PVDF membrane in 100% methanol for
5 min each.
Caution: Methanol is a very volatile and toxic solvent. It
should be handled in a fume hood and all the required steps
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
183
to wash the PVDF membrane should be performed in a fume
hood.
Incubate the PVDF membrane twice in PBST for 5 min each time
to equilibrate it for successive immunodecoration steps.
Block non-specific binding sites on the PVDF membrane by
equilibrating it in 5% (w/v) non-fat dry milk in PBST for at least
60 min.
Wash the membrane thrice with abundant PBST for 10 min
each.
Incubate the membrane in primary antibody solution for 2 h.
Note: Primary antibody solution is prepared by diluting
the primary antibody in 5% (w/v) non-fat dry milk in PBST.
Antibody dilution is highly variable; for instance, we use antidinitrophenyl-KLH antibodies, rabbit IgG fraction (cod. A6430)
from Molecular Probes at a 1:10,000 dilution (in combination with secondary goat anti-rabbit IgG, HRP conjugate (cod.
G21234) from Molecular Probes), which is sufficient to develop
a signal in ECL within 15 min. If a particularly weak signal is
expected, overnight primary antibody incubation at 4 ◦ C, with
gentle agitation, is the better choice to improve ECL signal.
Note: Use antibody solution only once and prepare it just
before use.
Wash the membrane thrice with abundant PBST for 10 min
each.
Incubate the membrane with secondary antibody at RT for at
least 1 h. We use secondary goat anti-rabbit IgG, HRP conjugate (cod. G21234) from Molecular Probes at a 1:2,000 dilution,
which is sufficient to develop a signal in ECL within 15 min (in
combination with primary rabbit IgG fraction (cod. A6430) from
Molecular Probes).
Note: Use antibody solution only once and prepare it just
before use.
Wash the membrane thrice with abundant PBST for 10 min
each.
Cover the membrane with ECL working solution according to
ECL manufacturer’s instruction. In general, depending on the
ECL kit, 1 to 5 min is enough time for the reaction to develop.
Discard excess of solution, wipe the back of the membrane with
a paper towel and wrap the membrane between two plastic
sheets.
Be sure that no air bubbles are trapped between the membrane and plastic wrap on the side to be exposed to X-ray film;
proceed immediately to expose the membrane to X-ray film.
Positive and negative controls are highly recommended in
order to check, respectively, proper reactivity of anti-DNP antibodies and to exclude any non-specific reactivity not due to
DNPH binding to PCO.
After ECL development, disassemble the plastic sandwich and
rinse abundantly the membrane in PBST in order to wash away
all ECL reagents.
(Advice: We suggest washing the membrane for at least 1 h
with frequent changes of PBST).
Proceed to stain the membrane with Amido Black (see Supplementary for Amido Black stain solution and Amido Black destain
solution).
Caution: Methanol is toxic and acetic acid is corrosive; these
solvents should be handled in a fume hood. Preparation of
staining and destaining solutions for Amido Black should be
performed in a fume hood and also all the steps for staining
(from 16 to 20) should be performed in a fume hood.
Incubate the membrane for 5 min in Amido Black stain solution.
Transfer the membrane in Amido Black destain solution and
rinse for 5 min.
Change the Amido Black destain solution every 5 min and proceed until the membrane background is white (Fig. 3B).
184
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
23. Lay the membrane on a paper towel under a fume hood and let
it air dry.
2.6. Immunoblot detection of PCO after 2D SDS-PAGE separation
Two-dimensional gel electrophoresis (2D-GE) is a protein separation technique that takes advantage of two physical protein
properties, isoelectric point and molecular mass, to allow the separation of a complex mixture of polypeptides in a 2D map. 2D-GE
employs a first isoelectrofocusing separation step, in which proteins are separated according to their isoelectric point, and a
second classical SDS-PAGE that separates proteins according to
their molecular mass. The result is a 2D map in which a single protein isoform appears as a spot on a 2D plane. It is basically like to
imagine a point on the Cartesian plane knowing that the x and y
coordinates define the location on the plane. Similarly, for a protein
isoform spot, after separation on a 2D map, its coordinates x and y
will be the isoelectric point and molecular mass. Considering that
it is unlikely that two polypeptide chains have the same isoelectric point and molecular mass, they are more effectively separated
in 2D-GE than in SDS-PAGE (and, in general, in 1D electrophoresis). 2D-GE is the preliminary step in case identification of specific
carbonylated proteins by mass spectrometry is required. In fact, a
single spot can be cut out of the gel and processed to be identified by Matrix-Assisted Laser Desorption/Ionization Time of Flight
(MALDI-TOF) mass spectrometry.
The following protocol can be used to separate both nonderivatized proteins and DNPH-derivatized proteins (see Section
2.3). In the first case, proteins are precipitated (e.g., by means of
chloroform/methanol protein precipitation described in Section
2.6.1), resuspended in isoelectrofocusing appropriate buffer (UTC
solution, see Supplementary), separated by 2D-GE, transferred to a
PVDF membrane and immunodetected by indirect on-membrane
protein labelling with DNPH (see Section 2.5). In the second case,
DNPH-derivatized protein pellets (see Step 11, Section 2.3.2) can
be directly resuspended in isoelectrofocusing appropriate buffer
(UTC solution), (see Supplementary and Step 12, Section 2.6.1.2),
separated by 2D-GE, transferred to a PVDF membrane and immunodetected (see from Step 3, Section 2.4.2).
2.6.1. Chloroform/methanol protein precipitation
Note: Protein precipitation can be obtained following various
protocols different from this one. We have used chloroform/methanol precipitation because it showed the highest
efficiency of precipitation with our protein samples and because
it also has the advantage to allow elimination of solvents by simple
evaporation under a fume hood.
2.6.1.1. Required chemicals.
•
•
•
•
1 mg/ml protein solution
chloroform
methanol
milliQ water
2.6.1.2. Procedure.
1. Starting from a protein solution at a concentration higher than
1 mg/ml, collect in a new test tube a volume corresponding
to the amount of proteins required; in general, 50–100 ␮g of
proteins are sufficient both for immunodetection of blotted
proteins and for spot cutting from 2D polyacrylamide gel and
protein identification by MALDI-TOF mass spectrometry.
(Advice: Too diluted protein solutions can result in a low precipitation efficiency).
2. Add 4 volumes of methanol and vortex-mix few seconds.
3. Add 1 volume of chloroform and vortex-mix few seconds.
4. Add 3 volumes of milliQ water and vortex-mix few seconds.
Note: Protein precipitation is suddenly evident; as water is
added, the solution becomes milky due to protein precipitation.
5. Centrifuge at 10,000 × g for 10 min at 4 ◦ C to collect the protein
pellet.
Note: After centrifugation, sample should appear as a twophase solution and, between the aqueous (upper) and organic
(lower) phase, a white thin protein layer should be visible.
6. Remove the upper aqueous phase paying attention not to perturb the protein layer.
7. Add 4 volumes of methanol and vortex-mix few seconds.
8. Centrifuge at 10,000 × g for 10 min at 4 ◦ C to collect the protein
pellet.
Note: After centrifugation, the protein pellet should be visible on the bottom of the test tube and no separation between
phases should appear.
9. Remove the supernatant paying attention not to perturb the
protein pellet.
10. Centrifuge at 10,000 × g for 1 min at 4 ◦ C to collect residual
supernatant and remove it.
11. Dry the pellet to eliminate any chloroform and methanol
residue.
(Advice: Air-drying, vacuum pump or Savant DNA SpeedVac®
Concentrators are allowed. We suggest constantly checking
the pellet during drying because overdrying could make pellet
resuspension hard to obtain, thus causing loss of proteins).
12. Resuspend the dried pellet in protein resuspension solution for
isoelectrofocusing (UTC solution, see Supplementary).
Depending on the preferred protocol, proteins can be in-solution
derivatized with DNPH (see Section 2.3.2) and subsequently resuspended in UTC solution; alternatively, non-derivatized proteins can
simply be precipitated (see Section 2.6.1.2, Steps 1–10) and directly
resuspend in UTC solution.
2.6.2. First dimension separation: isoelectric focusing with Ettan
IPGphor II system and strip holder
2.6.2.1. Required chemicals and equipment.
•
•
•
•
•
•
Ettan IPGphor II System (GE Healthcare)
Protein sample in UTC solution (see Supplementary)
Immobilized pH gradient (IPG) strips
Strip holder of appropriate length (e.g., 11 cm)
IPG buffer (e.g., IPG Buffer, pH 3–10 linear gradient)
Cover fluid oil
Note: IPG strips are commercially available in different pH
ranges and lengths. The choice of the pH range depends on the
aim of the 2D-GE analysis (global view of the sample proteome or
isolation of a specific protein isoform) and on the isoelectric point
of the protein of interest (e.g., 11 cm, pH 3–10 linear gradient IPG
strips; GE Healthcare).
Note: IPG buffers are ampholyte-containing buffers specifically
formulated for use with Immobiline DryStrip gels. Each IPG buffer
type produces more uniform conductivity along the Immobiline
DryStrip during focusing.
2.6.2.2. Procedure.
1. Starting from a protein sample in UTC solution, add IPG buffer
to a final concentration of 0.5–2%, vortex-mix and centrifuge at
10,000 × g for 1 min to collect the protein sample.
Note: The final concentration of IPG buffer depends on protein
sample and should be determined empirically. In general, in our
hands, a final concentration of 1% IPG buffer in protein sample
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
2.
3.
4.
5.
was sufficient to obtain a good focalization of the majority of
the analyzed samples (e.g., cell lysates, tissue extracts, purified
proteins).
Take a strip holder and apply sample on the bottom of it; distribute the sample throughout the length of the strip holder
between the two electrodes.
Remove protective plastic sheet from Immobiline DryStrip and
apply it on the sample with the gel upside down.
(Advice: Pay attention not to have air bubbles between sample
in UTC and the Immobiline Drystrip because they could interfere
with a homogeneous current flow).
Cover the Immobiline Drystrip with 1–2 ml of mineral oil and
close strip holder with its cover.
Place strip holders on Ettan IPGphor electrode plate and run the
isoelectric focusing (IEF) program.
Note: A typical program for IEF separation is characterized by a
first step of passive (without current flow) rehydration, a second
step of active (with current flow) rehydration, a third step of
low-current focalization (in which proteins start to move inside
the strip) and a fourth step of high-current active focalization
(proteins focalize and reach their isoelectric point).
A typical program protocol for IEF is the following:
Duration Current Current
profile
Passive rehydration 1 h
No
–
Active rehydration 12 h
Yes
Constant
(I step)
Yes
Increasing
Active rehydration 0.5 h
(II step)
1h
Yes
Constant
Low-current
(I step)
1h
Low-current
Yes
Increasing
(II step)
1–2.5 h
High-current
Yes
Constant
(I step)
1–2.5 h
High-current
Yes
Increasing
(II step)
3–6 h
Yes
Constant
High-current
(III step)
Voltage
0V
30 V
from 30 to 500 V
500 V
from 500 to 2500 V
2500 V
from 2500 to 8000 V
8000 V
185
• Gel electrophoresis unit and transfer unit (see Supplementary for
gel formulation and transfer buffer)
• Cleland’s reagent or dithitreithol (DTT)
• Iodoacetamide (IAM)
• 0.5% (w/v) boiling agarose melted in 2× running buffer for SDSPAGE separation (see Supplementary for 5× running buffer)
2.6.3.2. Procedure.
1. Thaw the strip at RT in case of −80 ◦ C storage.
2. Incubate the strip for 15 min in equilibration buffer (see Supplementary) added with 10 mg/ml DTT to reduce protein disulphide
bonds.
3. Rinse the strip with milliQ water and incubate it for 15 min in
equilibration buffer (see Supplementary) added with 25 mg/ml
IAM to alkylate reduced protein sulfhydryl groups.
(Advice: Alkylation is warmly recommended because free
sulfhydryl groups can cause vertical protein streaking during the
second-dimension separation).
4. Rinse the strip with milliQ water and dry from excess water on
a paper towel.
5. Place the strip on top of a 12.5% polyacrylamide gel and fix the
strip pouring a sufficient volume of 0.5% (w/v) boiling agarose in
2× running buffer.
(Advice: Pay attention not to have air bubbles between strip
and polyacrylamide gel surface because they could interfere with
current run and lead to deformation in protein separation).
6. Run SDS-PAGE according to manufacturer’s instruction.
7. After SDS-PAGE, the gel can be used to transfer proteins to PVDF
membrane as for 1D SDS-PAGE.
8. In case of 2D separation of DNPH-derivatized proteins, the
PVDF membrane can be processed according to “Immunoblot
detection of PCO” (see Section 2.4). Otherwise, in case of not
DNPH-derivatized proteins, the PVDF membrane has to be processed according to “Indirect on-membrane protein labelling
with DNPH” as described in Section 2.5 (Figs. 4 and 5).
2.7. PCO labelling with hydrazide
6. After IEF, strips are rinsed with milliQ water and it is possible to
proceed immediately to second dimension protocol. Otherwise,
strips can be stored at −80 ◦ C until 2D-GE will be performed.
2.6.3. Second dimension separation
2.6.3.1. Required chemicals and equipment.
Hydrazide (HYD) and its X-tagged hydrazide (e.g., biotinhydrazide, BHZ) are molecules able to react with carbonyl groups to
form the corresponding Schiff bases. Considering that these adducts
are unstable, they are usually reduced to more stable amines using
sodium cyanoborohydride to reduce double bond between carbon
and nitrogen (Fig. 6).
• Equilibration buffer for 2D-GE (see Supplementary)
2.7.1. PCO labelling with biotin-hydrazide (BHZ)
Fig. 4. 2D-GE separation and immunodetection of carbonylated protein. Cigarette smoke-induced formation of PCO in HGFs. Protein carbonylation in whole-cell lysates
from HGFs exposed to 0 and 5 cigarette puffs for 1 min was detected using a Western blot assay including on-membrane carbonyl derivatization with DNPH, detection of
DNP-protein adducts by anti-DNP antibodies, and visualization of the immunoreactive protein spots by HRP-conjugated goat anti-rabbit IgG and ECL. Representative 2D
immunoblots corresponding to the detection of carbonylated proteins in homogenates of control HGFs (left panel) and HGFs exposed to five cigarette puffs (right panel) are
shown. MALDI-TOF mass spectrometry identified proteins are labelled with numbers. Reprinted with permission from [15]. Copyright© 2013 Elsevier Ireland Ltd.
186
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
Fig. 5. 2D-GE separation and immunodetection of carbonylated protein. Redox proteomic analysis of CSE-induced PCO formation in human umbilical vein endothelial
cell line (ECV-304). Protein carbonylation was evaluated by means of Western blot
analysis with rabbit anti-DNP antibody, after on-membrane protein derivatization
with DNPH, and detected with HRP-conjugated goat anti-rabbit IgG and ECL. Representative 2D Western blots corresponding to the detection of carbonylated proteins
in homogenates of (A) control ECV-304 cells and ECV-304 cells exposed for 1 h to
2.5% (B), 5% (C), or 10% (D) CSE are shown. Reprinted with permission from [27].
Copyright© 2013 Elsevier Ireland Ltd.
2.7.1.1. Required chemicals.
•
•
•
•
•
•
Protein solution at a concentration higher than 2 mg/ml
BHZ derivatization buffer (see Supplementary)
20% (v/v) TCA solution, ice-cold (see Supplementary)
1:1 (v/v) EtOH:EtAc
30 mM BHZ in dimethyl sulfoxide (DMSO) (see Supplementary)
30 mM sodium cyanoborohydride (NaBH3 CN) (see Supplementary)
• 50 mM PBS, pH 7.4 (see Supplementary)
• 100% TCA (see Supplementary)
1. Dilute protein sample directly in BHZ derivatization buffer to
have a final protein concentration of 1–2 mg/ml.
(Advice: According to our experience, we suggest to start the
protocol with a sample protein concentration of 1–2 mg/ml
because, after derivatization and protein precipitation, higher
protein concentrations could lead to formation of insoluble
pellets difficult to resuspend, whereas lower protein concentrations could result in a very reduced efficiency of protein
precipitation and subsequent protein recovery).
(Advice: Avoid Tris or other primary amine-containing buffers
during sample processing because these buffers react with
aldehydes and will quench the reaction with hydrazides).
2. Incubate the protein dilution at RT for at least 15 min.
3. Centrifuge at 10,000 × g for 15 min to eliminate eventual insoluble matter and use the supernatant.
4. Add BHZ in DMSO to a final concentration of 5 mM (e.g., 100 ␮l
of protein sample at 2 mg/ml in BHZ derivatization buffer
requires the addition of 20 ␮l of 30 mM BHZ in DMSO).
5. Incubate the mixture for 3 h in constant mixing at RT.
6. Incubate the reaction on ice for 15 min.
7. Add a volume of 30 mM sodium NaBH3 CN dissolved in 50 mM
PBS, pH 7.4.
Note: As shown in Fig. 6, NaBH3 CN is used to stabilize
the hydrazone bond formed between hydrazide and carbonyl
group.
8. Incubate on ice for 30 min shaking every 10 min.
9. Precipitate proteins adding a final concentration of 10% TCA.
10. Incubate on ice for 15 min.
11. Centrifuge for 10 min at 10,000 × g at 4 ◦ C to pellet BHZderivatized proteins.
12. Wash the pellet thrice with 1:1 (v/v) EtOH:EtAc.
13. Remove the supernatant paying attention not to perturb the
protein pellet.
14. Centrifuge at 10,000 × g for 1 min at 4 ◦ C to collect residual
supernatant and remove it.
15. Air-dry the pellet to eliminate any ethanol and ethylacetate
residue.
16. Depending on the subsequent protocol that must be applied, it
is possible to resuspend the dried protein pellet in:
2.7.1.2. Procedure.
Fig. 6. Biotin hydrazide reaction with carbonyl species (e.g., protein Michael adducts of acrolein). Protein-bound aldehyde adduct is labelled with BHZ and the hydrazone
bond is stabilized by reduction with NaCNBH3 . Modified from [28]. Copyright© 2013 Elsevier Ireland Ltd.
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
187
Fig. 7. Labelling of PCO with ARP and Western blot detection with NeutrAvidin. SDS-PAGE (A) and Western blot (B) of interfibrillar heart mitochondria proteins from young
and old rats after reaction with ARP, with detection of biotinylated proteins by affinity staining with NeutrAvidin. M, biotinylated SDS-PAGE standard; CON, not labelled with
ARP; ARP = labelled with ARP. Identified proteins by MALDI-TOF MS/MS: 1, aconitate hydratase; 2, myosin; 3, ATP synthase ␣ subunit; 4, ATP synthase ␤ subunit; 5, aspartate
aminotransferase; 6, ADP/ATP translocase 1; 7, cytochrome c oxidase subunit IV. Reprinted with permission from [29]. Copyright© 2013 Elsevier Ireland Ltd.
a 1× Laemmli sample buffer (see Supplementary) for subsequent
analysis by 1D SDS PAGE, transfer to PVDF membrane and
immunodetection of PCO by HRP-conjugated streptavidin.
b Protein resuspension solution for IEF (UTC) (see Supplementary)
for subsequent analysis by 2D-GE separation, transfer to PVDF
membrane and immunodetection of PCO by HRP-conjugated
streptavidin.
2.7.2. Western blot detection of PCO after labelling with BHZ
After PCO derivatization with BHZ (see Section 2.7.1), derivatized proteins can be separated by 1D/2D SDS-PAGE as described
in Sections 2.4 and 2.6. Once the proteins are electrotransferred
to a PVDF membrane, it is possible to proceed with a single-step
detection protocol employing HRP-conjugated streptavidin (e.g.,
streptavidin-HRP (cod. RPN1231V) from GE Healthcare).
2.
3.
4.
5.
6.
2.7.2.1. Required chemicals and equipment.
• PVDF membrane from 1D or 2D Western blotting with BHZderivatized proteins
• PBS (pH 7.2) with 0.1% Tween-20 (PBST) (see Supplementary)
• 5% (w/v) non-fat dry milk in PBST
• HRP-conjugated streptavidin (streptavidin-HRP)
• ECL Western blotting detection reagents (e.g., from GE Healthcare)
• Plastic wrap, 100 ␮m thick
• X-ray film
7.
8.
9.
10.
2.7.2.2. Procedure.
1. After protein transfer to a PVDF membrane, wash the membrane for 15 min in PBST and continue with immunodecoration.
(Advice: In case the PVDF membrane must be stored for long
periods of time, after electroblotting, wash the membrane for
5 min in methanol, let it air dry and store it at −20 ◦ C until
use. To restart the immunodecoration protocol, equilibrate the
11.
PVDF membrane in PBST for at least 60 min in order to allow a
complete rehydration and continue with step 3).
Block the non-specific binding of the membrane equilibrating
it in 5% (w/v) nonfat dry milk in PBST for at least 60 min.
Wash the membrane thrice with abundant PBST solution for
10 min each.
Incubate the membrane in streptavidin-HRP solution for 2 h.
Note: The solution is prepared by diluting streptavidin-HRP
in 5% (w/v) non-fat dry milk in PBST. Streptavidin-HRP dilution is variable; for instance, we use streptavidin-HRP (cod.
RPN1231 V) from GE Healthcare at a 1:5,000 dilution, which
is sufficient to develop a signal in ECL within 10 min.
Note: Use streptavidin-HRP solution only once.
Wash the membrane thrice with abundant PBST for 10 min
each.
Cover the membrane with ECL working solution according to
ECL manufacturer’s instruction. In general, depending on the
ECL kit, 1–5 min is enough time for the reaction to develop.
Discard excess of solution and wrap the membrane between
two plastic sheets.
Be sure that no air bubbles are trapped between the membrane and plastic wrap on the side to be exposed to X-ray film;
proceed immediately to expose the membrane to X-ray film.
Positive and negative controls are highly recommended in
order to check, respectively, proper reactivity of streptavidinHRP and to exclude any non-specific reactivity not due to BHZ
binding to PCO.
After ECL development, disassemble the plastic sandwich and
rinse abundantly the membrane in PBST in order to wash away
all ECL reagents.
(Advice: We suggest washing the membrane for at least 1 h
with frequent changes of PBST).
Proceed to stain the membrane with Amido Black (see Supplementary for Amido Black stain solution and Amido Black destain
solution) as described in Section 2.5.2 from Step 19.
188
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
Fig. 8. 2D-GE profile of FTC-labelled oxidized and unoxidized liver proteins. Cytosolic proteins were oxidized with FeSO4 (12 mM) and ascorbic acid (3 mM). Proteins (100 ␮g)
were resolved by 2D-GE and the images show the fluorescence of FTC binding to the unoxidized (A) and oxidized protein extracts (B), respectively. (C, D) Images of the Sypro
Ruby fluorescence of the identical gels shown in panels A and B, respectively. (E, F) Images of the Sypro Ruby fluorescence of the unoxidized (E) and oxidized (F) proteins in
gels. Reprinted with permission from [31]. Copyright © 2013 Elsevier Ireland Ltd.
2.8. PCO labelling with aldehyde-reactive probe (ARP)
(O-(biotinylcarbazoylmethyl) hydroxylamine)
ARP is a molecule synthesized by reacting BHZ with O(carboxymethyl) hydroxylamine in the presence of carbodiimide. It
is applied to protein carbonylation as an alternative to hydrazidebased reagents because the derivatives are more stable than
hydrazones and do not require the additional reduction step with
NaBH3 CN. The ARP probe reacts specifically with aldehyde groups
resulting from protein oxidative modifications. Labelling with ARP
leads aldehyde sites in proteins to be converted to biotin-tagged
aldehyde sites, which can be detected using avidin-conjugated
probes (Fig. 7). We have rarely used this molecule in our lab, but a
useful paper employed ARP in Western blot analysis [30].
2.8.1. Required chemicals
• Protein solution (at least 50-100 ␮g)
• 25 mM ARP in DMSO (see Supplementary)
• 10 mM PBS, pH 7.4 (see Supplementary)
2.8.2. Procedure
1. Dilute 100 ␮g of protein sample directly in 10 mM PBS, pH 7.4.
2. Incubate the protein solution at RT for at least 15 min.
3. Centrifuge at 10,000 × g for 15 min to eliminate eventual insoluble matter and use the supernatant.
4. Add ARP in DMSO to a final concentration of 5 mM. (e.g., 100 ␮l
of 2 mg/ml protein sample requires the addition of 25 ␮l of
25 mM ARP in DMSO).
5. Incubate the mixture for 1 h in constant mixing at 37 ◦ C.
6. Add a volume of 2× Laemmli sample buffer (see Supplementary).
7. Heat samples at ∼95 ◦ C for 5 min.
8. Separate 10–20 ␮g of ARP-labelled proteins by SDS-PAGE in
a minigel electrophoresis unit according to manufacturer’s
instructions.
9. Transfer the electrophoresed proteins from the SDS-PAGE gel
onto a PVDF membrane by electroblotting using a transfer unit
according to manufacturer’s instructions.
10. Probe the blot for PCO with streptavidin-HRP as described in
Section 2.7.2.
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
2.9. Fluorescent probes for in-gel PCO detection
Although thus far few research papers have been published on
the use of fluorescent probes for detection of carbonylated proteins
[31,32], these molecules represent a valid possibility for studies of
protein carbonylation, because they show many advantages compared to the carbonyl-specific molecules described previously in
this chapter (see Sections 2.3, 2.7 and 2.8):
1) In general, fluorescent signal has a higher sensitivity than a nonfluorescent one.
2) Labelling protocols are similar to those for PCO labelling with
non-fluorescent probes, but the fluorescent signal detection
can be directly performed on-gel without performing Western
blot followed by immunodetection, therefore saving time and
resources.
3) In general, non-fluorescent molecules require the run of two
identical gels: one for protein staining and a second for protein
transfer to a PVDF membrane. In case of fluorescent probes, the
same gel used for acquisition of fluorescence signal can immediately be used to stain proteins and isolate protein bands/spots.
This advantage excludes the possibility of mismatch between
proteins on the gel and proteins on the membrane.
2.9.1. PCO labelling with fluorescein-5-thiosemicarbazide (FTC)
2.9.1.1. Required chemicals.
• Protein solution at a concentration higher than 2 mg/ml
• 20 mM FTC in dimethyl formammide (DMF) (see Supplementary)
2.9.1.2. Procedure.
1. Dilute protein sample to a final concentration of 1 mg/ml.
2. Add FTC in DMF to a final concentration of 1 mM.
3. Incubate the mixture for 2–3 h in constant mixing in the dark
at RT.
4. Precipitate proteins adding a volume of ice-cold 20% TCA (see
Supplementary).
5. Incubate on ice for 15 min.
6. Centrifuge for 10 min at 10,000 × g at 4 ◦ C to pellet FTCderivatized proteins.
7. Wash the pellet 5 times with 1:1 (v/v) EtOH:EtAc.
8. Remove the supernatant paying attention not to perturb the
protein pellet.
9. Centrifuge at 10,000 × g for 1 min at 4 ◦ C to collect residual
supernatant and remove it.
10. Air-dry the pellet to eliminate any ethanol and ethyl acetate
residue.
11. Depending on the subsequent protocol that must be applied, it
is possible to resuspend the dried protein pellet in:
a.) 1× Laemmli sample buffer (see Supplementary) for subsequent analysis by 1D SDS-PAGE.
b.) Protein resuspension solution for IEF (UTC) (see Supplementary) for subsequent analysis by 2D SDS-PAGE
separation
12. Proceed to 1D or 2D SDS-PAGE separation as described above.
After electrophoresis, the 1D or 2D gel is transferred to a fluorescence imager (e.g., Typhoon 9400 GE Healthcare) and the
image of FTC-labelled proteins is captured using an excitation
wavelength of 488 nm and an emission filter at 520 nm (Fig. 8).
3. Conclusions
In this protocol paper, we described the most easy-to-use and
accessible procedures to assess protein carbonylation in biolog-
189
ical samples. All the reported protocols require instrumentation
usually available in a standard research laboratory devoted to
proteome analysis (e.g., spectrophotometer, SDS-PAGE apparatus,
2D-GE and Western blot equipment). In particular, immunodetection of DNPH-derivatized carbonylated proteins can be used
with high flexibility according to the available equipment and,
if necessary, it can be coupled with dot blot, 1D or 2D SDSPAGE separation [14,15,26]. We are aware that this is not a
comprehensive overview of methods for measuring protein carbonylation. As clearly reported in literature, many other strategies
can be applied, especially considering the possibility to employ
more expensive and less conventional instrumentation, such as
high performance liquid chromatography (HPLC) following PCO
derivatization with DNPH [11,12], ELISA techniques [16,17], fluorescence microplate reader following PCO derivatization with
7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH), which form
highly fluorescent derivatives with carbonyl groups [33] or the
most expensive mass spectrometry [34–36], as investigation tools.
In addition, the combination of multiple techniques for PCO
detection was reported. For example, the coupling of BHZ with
avidin-FITC as labelling molecule and detection probe, respectively [37], allows flexibility in the protein carbonylation study and
multiple choice according to available instrumentation. Recently,
the chemical synthesis of new fluorophore-coupled hydrazide
molecules [38–40] has demonstrated usefulness and efficiency and,
consequently, specific PCO labelling tools are rapidly increasing.
A useful review clearly describes advantages, disadvantages and
applicability of many chemical probes for protein carbonylation
study [41]. In our opinion, its reading can be a fundamental step
to select the most suitable labelling molecule according to your
starting biological system and research requirements. In addition,
a useful review describes in details sample preparation for the
analysis of protein carbonylation with the aim to prevent possible
biological oxidation and artifactual events during protein extract
preparation [25].
Carbonylation of proteins has received a lot of attention in the
last decades due to the fact that it has been shown to accumulate
and to be implicated in the progression and the pathophysiology of
several diseases such as Alzheimer’s disease and coronary heart diseases [42,43]. The development of specific labelling molecules, the
standardization of methods, the reduction of interfering molecules
will be necessary to measure more accurately this oxidative protein modification. Consequently, a more accurate determination
of carbonylated protein level and species will help in understanding more extensively the role of protein carbonylation in biological
homeostasis and shedding light on molecular mechanisms underlying related pathologies.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jchromb.2015.
11.052.
References
[1] E.R. Stadtman, Metal ion-catalyzed oxidation of proteins: biochemical
mechanism and biological consequences, Free Radic. Biol. Med. 9 (1990)
315–325.
[2] I.M. Møller, A. Rogowska-Wrzesinska, R.S.P. Rao, Protein carbonylation and
metal-catalyzed protein oxidation in a cellular perspective, J. Proteomics 74
(2011) 2228–2242, http://dx.doi.org/10.1016/j.jprot.2011.05.004.
[3] P. Grimsrud, H. Xie, T.J. Griffin, D.A. Bernlohr, Oxidative stress and covalent
modification of protein with bioactive aldehydes, J. Biol. Chem. 283 (2008)
21837–21841, http://dx.doi.org/10.1074/jbc.R700019200.
[4] O. Jung, S. Min, R.A. Floyd, J. Park, Thiol-dependent metal-catalyzed oxidation
of copper, zinc superoxide dismutase, Biochim. Biophys. Acta 1387 (1998)
249–256.
190
G. Colombo et al. / J. Chromatogr. B 1019 (2016) 178–190
[5] H.N. Saada, U.Z. Said, E.M.E. Mahdy, H.E. Elmezayen, S.M. Shedid, Fish oil
omega-3 fatty acids reduce the severity of radiation-induced oxidative stress
in the rat brain, Int. J. Radiat. Biol. 90 (2014) 1179–1183, http://dx.doi.org/10.
3109/09553002.2014.934928.
[6] J.R. Requena, R.L. Levine, C. Chao, E.R. Stadtman, Glutamic and aminoadipic
semialdehydes are the main carbonyl products of metal-catalyzed oxidation
of proteins, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 69–74, http://dx.doi.org/10.
1073/pnas.011526698.
[7] H.M. Stringfellow, M.R. Jones, M.C. Green, A.K. Wilson, J.S. Francisco,
Selectivity in ROS-induced peptide backbone bond cleavage, J. Phys. Chem. A
118 (2014) 11399–11404, http://dx.doi.org/10.1021/jp508877m.
[8] M. Perluigi, R. Coccia, D.A. Butterfield, 4-Hydroxy-2-nonenal, a reactive
product of lipid peroxidation, and neurodegenerative diseases: a toxic
combination illuminated by redox proteomics studies, Antioxid. Redox Signal.
17 (2012) 1590–1609, http://dx.doi.org/10.1089/ars.2011.4406.
[9] P.J. Thornalley, N. Rabbani, Detection of oxidized and glycated proteins in
clinical samples using mass spectrometry—A user’s perspective, Biochim.
Biophys. Acta Gen. Subj. 1840 (2014) 818–829, http://dx.doi.org/10.1016/j.
bbagen.2013.03.025.
[10] R.L. Levine, D. Garland, C.N. Oliver, A. Amici, I. Climent, A.G. Lenz, B.W. Ahn, S.
Shaltiel, Determination of carbonyl content in oxidatively modified proteins,
Methods Enzym. 186 (1990) 464–478.
[11] S. Uchiyama, Y. Inaba, N. Kunugita, Derivatization of carbonyl compounds
with 2,4-dinitrophenylhydrazine and their subsequent determination by
high-performance liquid chromatography, J. Chromatogr. B 879 (2011)
1282–1289, http://dx.doi.org/10.1016/j.jchromb.2010.09.028.
[12] E. Cirera-Domènech, R. Estrada-Tejedor, F. Broto-Puig, J. Teixidó, M.
Gassiot-Matas, L. Comellas, et al., Quantitative structure-retention
relationships applied to liquid chromatography gradient elution method for
the determination of carbonyl-2,4-dinitrophenylhydrazone compounds, J.
Chromatogr. A 1276 (2013) 65–77, http://dx.doi.org/10.1016/j.chroma.2012.
12.027.
[13] H. Buss, T.P. Chan, K.B. Sluis, N.M. Domigan, C.C. Winterbourn, Protein
carbonyl measurement by a sensitive ELISA method, Free Radic. Biol. Med. 23
(1997) 361–366, http://dx.doi.org/10.1016/S0891-5849(97)00104-4.
[14] N.B. Wehr, R.L. Levine, Quantitation of protein carbonylation by dot blot, Anal.
Biochem. 423 (2012) 241–245, http://dx.doi.org/10.1016/j.ab.2012.01.031.
[15] G. Colombo, I. Dalle-Donne, M. Orioli, D. Giustarini, R. Rossi, M. Clerici, et al.,
Oxidative damage in human gingival fibroblasts exposed to cigarette smoke,
Free Radic. Biol. Med. 52 (2012) 1584–1596, http://dx.doi.org/10.1016/j.
freeradbiomed.2012.02.030.
[16] G. Caimi, E. Hopps, D. Noto, B. Canino, M. Montana, D. Lucido, et al., Protein
oxidation in a group of subjects with metabolic syndrome, Diabetes Metab.
Syndr. Clin. Res. Rev. 7 (2013) 38–41, http://dx.doi.org/10.1016/j.dsx.2013.02.
013.
[17] E. Augustyniak, A. Adam, K. Wojdyla, A. Rogowska-Wrzesinska, R. Willetts, A.
Korkmaz, et al., Validation of protein carbonyl measurement: a multi-centre
study, Redox Biol. 4 (2015) 149–157, http://dx.doi.org/10.1016/j.redox.2014.
12.014.
[18] B.A. Soreghan, F. Yang, S.N. Thomas, J. Hsu, High-throughput proteomic-based
identification of oxidatively induced protein carbonylation in mouse brain,
Pharm Res. 20 (2003) 1713–1720.
[19] M.R. Roe, H. Xie, S. Bandhakavi, T.J. Griffin, Proteomic mapping of
4-hydroxynonenal protein modification sites by solid-phase hydrazide
chemistry and mass spectrometry, Anal. Chem. 79 (2007) 3747–3756, http://
dx.doi.org/10.1021/ac0617971.
[20] B.C. Hollins, S.a. Soper, J. Feng, Enriching carbonylated proteins inside a
microchip through the use of oxalyldihydrazide as a crosslinker, Lab Chip. 12
(2012) 2526, http://dx.doi.org/10.1039/c2lc40103g.
[21] J. Renke, S. Popadiuk, M. Korzon, B. Bugajczyk, M. Wozniak, Protein carbonyl
groups content as a useful clinical marker of antioxidant barrier impairment
in plasma of children with juvenile chronic arthritis, Free Radic. Biol. Med. 29
(2000) 101–104.
[22] J. Himmelfarb, E. McMonagle, E. McMenamin, Plasma protein thiol oxidation
and carbonyl formation in chronic renal failure, Kidney Int. 58 (2000)
2571–2578.
[23] C.S. Mesquita, R. Oliveira, F. Bento, D. Geraldo, J.V. Rodrigues, J.C. Marcos,
Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for
quantification of carbonyls in oxidized proteins, Anal. Biochem. 458 (2014)
69–71, http://dx.doi.org/10.1016/j.ab.2014.04.034.
[24] S. Luo, N.B. Wehr, Protein carbonylation: avoiding pitfalls in the
2,4-dinitrophenylhydrazine assay, Redox Rep. Commun. Free Radic. Res. 14
(2009) 159–166, http://dx.doi.org/10.1179/135100009x392601.
[25] A. Rogowska-Wrzesinska, K. Wojdyla, O. Nedić, C.P. Baron, H.R. Griffiths,
Analysis of protein carbonylation-pitfalls and promise in commonly used
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
methods, Free Radic. Res. 48 (2014) 1145–1162, http://dx.doi.org/10.3109/
10715762.2014.944868.
I. Dalle-Donne, M. Carini, M. Orioli, G. Vistoli, L. Regazzoni, G. Colombo, et al.,
Protein carbonylation: 2,4-dinitrophenylhydrazine reacts with both
aldehydes/ketones and sulfenic acids, Free Radic. Biol. Med. 46 (2009)
1411–1419, http://dx.doi.org/10.1016/j.freeradbiomed.2009.02.024.
G. Colombo, G. Aldini, M. Orioli, D. Giustarini, R. Gornati, R. Rossi, et al.,
Water-Soluble alpha,beta-unsaturated aldehydes of cigarette smoke induce
carbonylation of human serum albumin, Antioxid. Redox Signal. 12 (2010)
349–364, http://dx.doi.org/10.1089/ars.2009.2806.
R. Gornati, G. Colombo, M. Clerici, F. Rossi, N. Gagliano, C. Riva, et al., Protein
carbonylation in human endothelial cells exposed to cigarette smoke extract,
Toxicol. Lett. 218 (2013) 118–128, http://dx.doi.org/10.1016/j.toxlet.2013.01.
023.
P.C. Spiess, B. Deng, R.J. Hondal, D.E. Matthews, A. van der Vliet, Proteomic
profiling of acrolein adducts in human lung epithelial cells, J. Proteomics 74
(2011) 2380–2394, http://dx.doi.org/10.1016/j.jprot.2011.05.039.
W.G. Chung, C.L. Miranda, C.S. Maier, Detection of carbonyl-modified proteins
in interfibrillar rat mitochondria using
N -aminooxymethylcarbonylhydrazino-D-biotin as an aldehyde/keto-reactive
probe in combination with Western blot analysis and tandem mass
spectrometry, Electrophoresis 29 (2008) 1317–1324, http://dx.doi.org/10.
1002/elps.200700606.
A.R. Chaudhuri, E.M. de Waal, A. Pierce, H. Van Remmen, W.F. Ward, A.
Richardson, Detection of protein carbonyls in aging liver tissue: A
fluorescence-based proteomic approach, Mech. Ageing Dev. 127 (2006)
849–861, http://dx.doi.org/10.1016/j.mad.2006.08.006.
H.F. Poon, L. Abdullah, J. Reed, S.M. Doore, C. Laird, V. Mathura, et al.,
Improving image analysis in 2DGE-based redox proteomics by labeling
protein carbonyl with fluorescent hydroxylamine, Biol. Proc. Online 9 (2007)
65–72, http://dx.doi.org/10.1251/bpo134.
P. Stocker, E. Ricquebourg, N. Vidal, C. Villard, D. Lafitte, L. Sellami, et al.,
Fluorimetric screening assay for protein carbonyl evaluation in biological
samples, Anal. Biochem. 482 (2015) 55–61, http://dx.doi.org/10.1016/j.ab.
2015.04.021.
M. Colzani, G. Aldini, M. Carini, Mass spectrometric approaches for the
identification and quantification of reactive carbonyl species protein adducts,
J. Proteomics 92 (2013) 28–50, http://dx.doi.org/10.1016/j.jprot.2013.03.030.
I. Milic, M. Fedorova, Derivatization and detection of small aliphatic and
lipid-bound carbonylated lipid peroxidation products by ESI–MS, Methods
Mol Biol. 1208 (2015) 3–20, http://dx.doi.org/10.1007/978-1-4939-1441-8 1.
A.G. Madian, F.E. Regnier, Proteomic identification of carbonylated proteins
and their oxidation sites, J. Proteome Res. 9 (2010) 3766–3780, http://dx.doi.
org/10.1021/pr1002609.
B.S. Yoo, F.E. Regnier, Proteomic analysis of carbonylated proteins in
two-dimensional gel electrophoresis using avidin-fluorescein affinity
staining, Electrophoresis 25 (2004) 1334–1341, http://dx.doi.org/10.1002/
elps.200405890.
J. Tamarit, A. de Hoogh, E. Obis, D. Alsina, E. Cabiscol, J. Ros, Analysis of
oxidative stress-induced protein carbonylation using fluorescent hydrazides,
J. Proteomics 75 (2012) 3778–3788, http://dx.doi.org/10.1016/j.jprot.2012.04.
046.
K. Mukherjee, T.I. Chio, D.L. Sackett, S.L. Bane, Detection of oxidative
stress-induced carbonylation in live mammalian cells, Free Radic. Biol. Med.
84 (2015) 11–21, http://dx.doi.org/10.1016/j.freeradbiomed.2015.03.011.
V. Vemula, Z. Ni, M. Fedorova, Fluorescence labeling of carbonylated lipids
and proteins in cells using coumarin-hydrazide, Redox Biol. 5 (2015)
195–204, http://dx.doi.org/10.1016/j.redox.2015.04.006.
L.J. Yan, M.J. Forster, Chemical probes for analysis of carbonylated proteins: a
review, J. Chromatogr. B 879 (2011) 1308–1315, http://dx.doi.org/10.1016/j.
jchromb.2010.08.004.
D.A. Butterfield, M. Perluigi, T. Reed, T. Muharib, C.P. Hughes, R.a.S. Robinson,
et al., Redox proteomics in selected neurodegenerative disorders: from its
infancy to future applications, Antioxid. Redox Signal. 17 (2012) 1610–1655,
http://dx.doi.org/10.1089/ars.2011.4109.
M. Valko, D. Leibfritz, J. Moncol, M.T.D. Cronin, M. Mazur, J. Telser, Free
radicals and antioxidants in normal physiological functions and human
disease, Int. J. Biochem. Cell Biol. 39 (2007) 44–84, http://dx.doi.org/10.1016/j.
biocel.2006.07.001.
P. Wang, S.R. Powell, Decreased sensitivity associated with an altered
formulation of a commercially available kit for detection of protein carbonyls,
Free Radic. Biol. Med. 49 (2010) 119–121, http://dx.doi.org/10.1016/j.
freeradbiomed.2010.03.005.