Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Summary

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Abstract

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

Introduction

Mental and neurological disorders represent 13% of the global burden of disease, new challenges like pathophysiological mechanisms, risk factors and prodromal biomarkers must be explored¹. In line with this objective, proteomics studies of the human brain become indispensable to uncover molecular pathways involved in processes like memory, behavior, emotions and neuronal plasticity for instance, not only for physiological but also for pathological conditions. Therefore, the use of animal models and more specifically the transgenic mice, brings a wide range of possibilities to mimic the etiology of human neurodegenerative disorders².

Proteomics approaches are nowadays available in order to accomplish these new perspectives in the neuroscience field. Two-dimensional gel electrophoresis (2DE) is a potent and likely simple method that enables to compare the proteome of a wide range of samples. Moreover, it is also a powerful method to isolate a protein from a complex mixture in order to identify and further analyze by mass spectrometry. This technique essentially consists in two successive steps: 1) protein separation according to their isoelectric point (pI) by isoelectrofocusing (IEF). More precisely, an electric potential is applied across the immobiline acrylamide strips among a pH gradient and then proteins will migrate and focus on a determined pI in function of their global net charge. 2) Isoelectrically focused proteins are denatured and negatively charged by the addition of sodium dodecyl sulfate (SDS), thereby proteins in their first structure are separated depending on their apparent molecular weight (MW) by SDS-PAGE³. These two distinct properties allow us to tackle a double value to go further in the study of the proteome. On one hand this approach offers the possibility to perform a quantitative analysis using minimal dyes 2D-DIGE method, and on the other hand a qualitative analysis by mini-2DE coupled to western blotting.

Quantitative analysis by 2D-DIGE bestows protein expression changes all over the sample proteome. Briefly, samples are labeled with three cyanines (Cy2, Cy3 and Cy5) emitting at three distinct wavelengths (blue, green and red). These fluor minimal dyes containing N-hydroxy succinimidyl ester group react with the ε-amino group of lysines residues of proteins resulting in covalent amide bonds⁴. Lysine residues are labeled only between 1-3% and thus preventing multiple labels addition per protein and major net charge modifications^5,6. Cy3 and Cy5 are often used to label two independent samples while Cy2 tags a mix of equal proportion of the samples to compare. The two main advantages are that all labeled samples are mixed and IEF and SDS-PAGE are carried out in one gel at once for each step, avoiding the inter variability among experiments due to gels comparison. Moreover, it presents a high detection threshold of around 1 femtomole of protein⁷. Gels are scanned and 2D software compares the 2D gel fluorescence images, where Cy2 serves as an internal standard allowing for identification of statistical differences among the spots for their posterior identification by mass spectrometry. The 2D analysis software using the internal standard achieves a fast detection of less than 10% of differences between samples with more than 95% of statistical confidence⁸.

Qualitative analysis by mini-2DE is a crucial step for protein characterization. The principle is the same as previously described for pI and MW separation, but in this case proteins are transferred from a small polyacrylamide gel to a membrane and immunoblotting is performed afterwards. While one dimension gel electrophoresis provides changes in protein expression for one or several protein epitopes in function of the antibody, the information of mini-2DE endows with two additional parameters. Firstly, the protein isovariants change in function of the pI, indicating that post-translational modifications might take place. Secondly, mass spectrometry identification may be indicative of plausible zymogens and catabolic products of proteins. Therefore, modifications observed by 2D-DIGE are likely indicative of the mechanisms underlying changes in global proteome profile. Alignments of immunoblots among several samples for the same protein epitope/s by mini-2DE may reflect acidity changes shedding light into post-translational variations slightly observed or even not by monodimensional immunoblotting^9,10. Moreover, this analysis informs about the potential cleavage sites due to the knowledge of the pI and MW of the metabolic residues^11,12.

The combination of these two techniques provides a complementary proteomics analysis. On one hand 2D-DIGE affords a type of differences allowing a precise isolation of polypeptides whose expression is different. These differences consist essentially into the appearance or disappearance of a spot or increase/decrease of intensity of a given one by software analysis of the fluorescent gels. However, these observations by themselves are unlikely to explain the nature of the modification observed. For these reasons, once the polypeptide is isolated and identified by mass spectrometry, the use of mini-2DE enables to precisely confirm 1) the identity of the protein isolated and 2) the nature of the difference: change in the isovariant/isoform level of expression, post-translational modifications and cleavage processes for instance. However, it is necessary to develop a starting lysis buffer both compatible with 2D-DIGE and with mini-2DE in order to limit the potential dispersions resulting from the use of extraction protocols that are very dissimilar.

In the present article, we described an adapted protocol for the preparation, extraction and performance of 2D-DIGE and mini-2DE techniques for brain proteins coming from human and mouse tissue.

Protocol

1. Homogenization and Total Protein Extraction from Human and Mouse Brain Tissue

1. Brain tissue homogenization. Homogenize the brain tissue 10% (w/v) in 8 M urea, 2 M thiourea and 1% w/v SDS buffer (UTS) using a potter glass (for human samples) or Teflon homogenizer (for mice samples). Sonicate at 60 Hz 30 pulses with a ultrasound generator applying 0.5 sec for total tissue disintegration^13,14.
   1. Determine protein concentration with Bradford assay, using BSA as standard. This UTS buffer extraction is totally compatible with one dimension SDS-PAGE as long as lysates are not over-heated to avoid carbamylation¹⁵.
   2. Add 1% (w/v) of Amberlite and incubate with gentle mixing during 10 min at room temperature. Thereafter filter onto 0.22 µm syringe filters and finally add the SDS to the urea/thiourea solution. This step reduces the amount of uric acid and isocyanic acid, which are in equilibrium with urea in solution and facilitate its degradation.
2. Chloroform/methanol protein precipitation. Precipitate between 100-150 µg of protein for 11 cm IPG Strips and 1-1.5 mg for 18 cm ones (independently on the pH range selected to perform the IEF).
   1. Perform all the steps on ice or at 4 °C. Cool chloroform and methanol at 4 °C for 30 min before starting the precipitation procedure.
   2. Add three volumes of methanol and one volume of chloroform to one volume of UTS lysate. Shake manually the mixture and add three volumes of cold water. Vortex during 1 min and pellet the proteins by centrifugation at 12,000 x g for 30 min at 4 °C.
   3. Discard the supernatant using a Pasteur pipette and add three volumes of cold MeOH. Vortex again and centrifuge at 12,000 x g during 30 min at 4 °C. Finally discard supernatant and dry the pellet under N₂.
3. Preparation of proteins for 2D analysis. Resuspend the protein-dried pellet in 2D-buffer (8 M urea, 2 M thiourea supplemented with 4% CHAPS). Wash the solution with Amberlite as indicated above and try to keep the proportion of 500 µg of protein per 200 µl of 2D buffer for 2D-DIGE. Note: 2D buffer can be stored at -80 °C or kept at 4 °C during one week (to prevent urea degradation).
   1. Sonicate and store the samples at -80 °C until carrying out the following step. Noteworthy, this protocol can be used after formic acid solubilization of protein aggregates¹⁰. Briefly, evaporate formic acid under N₂ and resuspend the pellet as described.

2. 2D-DIGE

1. Samples test quality. Dose samples again using Bradford method. Dilute 15 µg of proteins in LDS sample buffer, heat at 37 °C¹⁵ and load onto 4-12% polyacrylamide gels.
2. Coomassie staining. Stain polyacrylamide gel with 0.1% (w/v) Coomassie Blue G-250, 50% EtOH and 10% (v/v) acetic acid solution for at least 1 hr. Let background destain overnight in 7% acetic acid and 10% (v/v) ethanol solution.
3. Pre-electrophoretic labeling of samples. Prior to performing cyanine dye labeling, verify the pH of the sample, which should be basic or even neutral since the N-hydroxysuccinimide substitution is more efficient at basic pH and given to be optimal at pH 8.6.
   1. Label minimally the two samples of study with Cy3 or Cy5 fluorescent dyes with a ratio of 50 μg protein/400 pmol dye and keep for 1 hr at 4 °C in the darkness to facilitate the NHS ester reactive group of the cyanines with the unprotonated amine groups of lysines.
   2. Reduce sample variations making a cross-labeling with Cy3 and Cy5 dyes, avoiding a preferential coupling of one sample to a particular cyanine. Note that the cyanine-dye minimal labeling can be adapted for small amount of proteins with keeping the proportion of 1 µg protein per 8 pmol cyanine-dye. If so, the mini-2DE system can be used instead of the large 2D gel system. This will enable the 2D-DIGE analysis for a small amount of brain tissue.
   3. Label a pool of both samples with the same amount of protein (50 µg in total) with Cy2 fluorescent dye and used as internal standard. Use the same conditions as previously indicated.
   4. Complete to a total volume 350 μl with 2D buffer containing 1.1% Destreak reagent, 1.2% of IPG buffer and traces of Bromophenol Blue the total 150 µg of labeled proteins (50 µg of Cy2, 50 µg of Cy3 and 50 µg of Cy2). Perform this step in quadruplicate using four independent strips. Moreover, prepare two additional non-labeled strips (one for each sample) with 350-450 µg of protein for the preparative gels.
   5. Leave the six loaded strips covered with mineral oil overnight for passive rehydration.
4. Isoelectrofocusing. Carry out the IEF at 20 °C. For a pH 3-11NL, 18 cm strip the protocol is: maximum current setting is 50 μA/strip during 8 stages: 1) 150 V step for 1 hr, 2) 200 V step for 5 hr, 3) 500 V gradient for 2 hr, 4) 1,000 V gradient for 2 hr, 5) 2,000 V gradient for 2 hr, 6) 4,000 V gradient for 2 hr, 7) 8,000 V gradient for 2 hr and 8) for a total of 24,000 V·hr step. After IEF, dispatch the excess of mineral oil using chromatography paper and store the strips at -20 °C until the second dimension.
5. IPG Strips equilibration. Equilibrate strips in 6 M urea, 2% SDS, 30% Glycerol, 50 mM Tris-HCl pH 8.6 buffer with 1% of DTT during 15 min and after with 4.7% of iodoacetamide in the same buffer but without DTT.
6. Second dimension or SDS-PAGE electrophoresis. Make the six 12% SDS-PAGE gels (1.5 M Tris-HCl pH 8.6, 12% acrylamide/bisacrylamide, 0.1% (w/v) SDS, 0.072% (w/v) APS and 0.1% (v/v) TEMED) at the same time using a large 2D system, with four low-fluorescence glass plates for the strips with fluorescence samples and two other glass plates with 1.5 mm of thickness for preparative gels in order to excise the protein spots.
   1. Load the six IPG Strips on the top of the gels and over layered with 0.5% (w/v) of agarose. Running buffer is composed of 25 mM Tris, 192 mM glycine and 0.1% (w/v) SDS. Carry out the migration at 2.5 W/gel overnight with a refrigeration system set to 4 °C^16,17.
7. Fluorescence images acquisition and analysis. Use a Typhoon 9400 scanner to obtain the fluorescence gels images (12 images for one experiment). Scan Cy2, Cy3 and Cy5 images for each gel at 488/520 nm, 532/580 nm and 630/670 nm excitation/emission wavelengths, respectively. Use informatic software to analyze the images. Data are representative of the changes of spot volume associated with the distribution of the signal across the four gels among the two samples studied.
8. Coomassie staining for big gels. Fix the preparative gels in 50% (v/v) EtOH and 3% (v/v) orthophosphoric acid solution for at least 24 hr. Then after, wash twice with ultra pure water for 20 min. Perform coloration in 34% (v/v) MeOH, 17% (w/v) ammonium sulfate and 2% orthophosphoric acid for 1 hr before adding 0.1% (w/v) Coomassie Blue G-250. Keep the gels in coloration for at least 48 hr and decolorized in ultra pure water.
   1. Spots for MS identification. Once fluorescence images are analyzed, the software provides a list of spots whose expression is statistically different. Manually excise these spots from the preparative gels. This procedure requires practice and some manual dexterity in order to avoid keratin contamination. Then samples can be kept in water at -20 °C until sending to MS. Spot identification is perfectly compatible with Tandem mass spectrometry (MS/MS) and relevance of protein identities is judged according to the probability based Mowse score calculated with a p-value of 0.05 (p < 0.05)¹⁸.

3. Qualitative Mini-2DE Assay

1. Resuspend maximum 100 µg of protein sample in 2D buffer until a final volume of 200 µl for a 11 cm IPG strip. In case of overexpression system or purified protein, the starting amount can be lowered to 20 µg.
2. Add 1.1% (v/v) Destreak Reagent, 0.55% (v/v) IPG buffer and traces of Bromophenol Blue to a final volume of 200 µl. Then, vortex, make a fast spin and load into an IPG strip for passive rehydration (to bring the strip to its original thickness) overnight covered with mineral oil. Note: the proportion of IPG buffer is the same for all the interval of pH desired (pH 3-11, 4-7, 7-11, etc), but its composition varies in function of the pH range selected.
3. Isoelectrofocalisation. Perform IEF at 20 °C. The duration of the program depends on the pH interval selected. Noteworthy, parameters such as electroendosmosis may disturb the IEF profile and in these cases, optimization in electrofocalisation time is required¹⁹. Note: it is not necessarily a longer IEF that provides better results but shortening the time of IEF can also improve the resolution. After IEF, IPG strips can be stored at -20 °C for months.
4. IPG strips equilibration. Equilibrate the strips in a buffer containing 25 mM Tris-HCl pH 6.8, 20 mM DTT, 10% glycerol, 5% SDS, 0.05% Bromophenol Blue. Make three equilibration bathes for 15 min with changing the buffer solution between each bath.
5. SDS-PAGE. Layer the IPG strip onto a precast gel (IPG+1 well, 11 cm IPG strip) with a percentage of polyacrylamide chosen depending on the MW of the protein of interest. Then, blot the gel onto a nitrocellulose/PVDF membrane at 100 mV during 33 min.
6. Incubate overnight with the required antibody and visualize by peroxidase activity. Perform the alignments of immunoblots using unrelated polypeptides revealed by the secondary antibody. Reach a semi-quantitative analysis by densitometry of the volume and intensity for the traces/ spots with ImageJ software.

Results

Proteomics on brain tissue remains challenging since no ideal buffer exists to recover 100% of proteins, especially membrane-associated or cytoskeleton proteins. The first set of experiments were focused on the search for a suitable lysis buffer compatible with the two approaches and which enables to recover a large panel of protein. Thus, three lysis buffers were assayed to determine the most appropriate one. Firstly, it was used the common biochemical and molecular biology buffer Tris-HCl²⁰ at 10 mM with 1% ...

Discussion

Discovering pathological expression changes of proteins, search for biomarkers and the modulation of potential pathways for pharmacological targets are among the aims of the neuroproteomics approaches³⁰. Amid the emerging tools, the 2DE field adds a promising expectative. Nevertheless, a consensus should be reached in order to minimize variability and increase reproducibility in the experiments. In line of this idea a standardized protocol to perform 2D-DIGE (Figure 2) and the subsequent 2DE i...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CyDye DIGE FLUOR MIN KIT 5 nmol 1 * 5 Nmo	GE Healtcare	25-8010-65
Immobiline DryStrip	GE Healtcare		Reference in function of the pH interval/size
IPG Buffer, pH X-X	GE Healtcare		Reference in function of the pH interval desired
AMBERLITE IRN-150L MIXED BED RESIN 1	GE Healtcare	551797N
DRYSTRIP COVER FLUID IMMOBILINE 1 * 1 l	GE Healtcare	17-1335-01
KIT BOX IPG 1 * 1 KIT	GE Healtcare	28-9334-92	Plastic box to make the passive rehydration
MILLEX GS Filter	Millipore	SLGS033SB
Criterion XT Precast Gels 13.3 x 8.7 cm (W x L)	Bio-Rad		Reference in function of the MW to separate
IPGphor III Isoelectric Focusing Unit	GE Healtcare	11-0033-64
Ettan DALTsix Large Electrophoresis System	GE Healtcare	80-6485-08
OneTouch 2D Gel SpotPicker 1.5 mm	Gel Company	P2D1.5