A step-by-step protocol for assaying protein carbonylation in biological samples

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Journal of Chromatography B, xxx (2015) xxx–xxx

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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 xxx 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: [email protected] (G. Colombo).

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].

http://dx.doi.org/10.1016/j.jchromb.2015.11.052 1570-0232/© 2015 Elsevier B.V. All rights reserved.

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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 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 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).

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.

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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:

3

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 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 concentra-

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tions 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. 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. 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. 8. Wash the membrane thrice with abundant PBST for 10 min each. 9. 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).

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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.

10. 11.

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17. 18. 19. 20.

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. (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). Lay the membrane on a paper towel under a fume hood and let it air dry.

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.

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)

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• • • •

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.

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 to wash the PVDF membrane should be performed in a fume hood. 7. Incubate the PVDF membrane twice in PBST for 5 min each time to equilibrate it for successive immunodecoration steps. 8. 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. 9. Wash the membrane thrice with abundant PBST for 10 min each. 10. 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.

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19.

20. 21. 22. 23.

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). 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

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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.

9. 10. 11.

12.

7

Note: After centrifugation, the protein pellet should be visible on the bottom of the test tube and no separation between phases should appear. Remove the supernatant paying attention not to perturb the protein pellet. Centrifuge at 10,000 × g for 1 min at 4 ◦ C to collect residual supernatant and remove it. 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). 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

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: 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 was sufficient to obtain a good focalization of the majority of the analyzed samples (e.g., cell lysates, tissue extracts, purified proteins). 2. 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. 3. 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). 4. Cover the Immobiline Drystrip with 1–2 ml of mineral oil and close strip holder with its cover.

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5. 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 Low-current Yes Constant (I step) 1h Yes Increasing Low-current (II step) 1–2.5 h High-current Yes Constant (I step) 1–2.5 h Yes Increasing High-current (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

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. • Equilibration buffer for 2D-GE (see Supplementary) • 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)

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 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). 2.7.1. PCO labelling with biotin-hydrazide (BHZ) 2.7.1.1. Required chemicals.

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.

2.7.1.2. Procedure.

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)

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.

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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.

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.

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).

2. 3. 4.

5. 6. 7.

8. 9. 10. 11. 12.

(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). Incubate the protein dilution at RT for at least 15 min. Centrifuge at 10,000 × g for 15 min to eliminate eventual insoluble matter and use the supernatant. 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). Incubate the mixture for 3 h in constant mixing at RT. Incubate the reaction on ice for 15 min. 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. Incubate on ice for 30 min shaking every 10 min. Precipitate proteins adding a final concentration of 10% TCA. Incubate on ice for 15 min. Centrifuge for 10 min at 10,000 × g at 4 ◦ C to pellet BHZderivatized proteins. Wash the pellet thrice with 1:1 (v/v) EtOH:EtAc.

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.

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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: 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). 2.7.2.1. 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 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 PVDF membrane in PBST for at least 60 min in order to allow a complete rehydration and continue with step 3). 2. Block the non-specific binding of the membrane equilibrating it in 5% (w/v) nonfat dry milk in PBST for at least 60 min. 3. Wash the membrane thrice with abundant PBST solution for 10 min each. 4. 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. 5. Wash the membrane thrice with abundant PBST for 10 min each. 6. 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. 7. Discard excess of solution and wrap the membrane between two plastic sheets.

8. 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. 9. 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. 10. 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). 11. 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. 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. 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):

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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.

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:

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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 biological 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.

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