SAMPLE EXTRACTION TECHNIQUES FOR ENHANCED PROTEOMIC ANALYSIS OF RAT BRAIN TISSUE

SAMPLE EXTRACTION TECHNIQUES FOR ENHANCED PROTEOMIC ANALYSIS OF RAT BRAIN TISSUE

A. Soggiu1, S. Pisanu°, A. Crippa°, P. Devoto*, P. Roncada2

°Proteotech s.r.l., Parco Tecnologico della Sardegna, Pula (Cagliari)

¹Laboratorio Proteomica, Dipartimento Scienze Cliniche e Veterinarie, Università degli studi di Milano

² Istituto Sperimentale Italiano L. Spallanzani

* Dipartimento di Neuroscienze, Università degli Studi di Cagliari

Abstract.

High resolution two dimensional electrophoresis (2DE) remains the core technology for

resolving complex protein mixtures, prior to mass spectrometry characterization. Due to the

complexity of brain tissue, optimising protein separation techniques is fundamental to unravel

the function of brain proteins and their role in disease. So, brain tissue is a very good model to

improve resolution due to either hydrophobic then basic characteristic of proteins. It has been

described that for wide pH 3–10 gradients, the use of HED (Hydroxyethyl disulfide) could

also be advantageous; although not needed for reduction of streaking, it has been result in an

improved reproducibility of the basic part of the resulting 2-D maps. In this work it has been

applied HED to perform rat brain two dimensional electrophoresis. Analytical loads in

combination with anodic cup application or paper bridge application has been used to

compare methods to resolve brain extract. Image analysis indicate that paper bridge loading

combined with HED give very sharp image and a strong reduction of streaking and improved

separation and focusing of spots according to literature; statistical analysis derived from

image analysis shows a loss of spots ( about 20% ) compared with anodic cup loading.

Therefore the latter method coupled to HED is the best choice for semi-quantitative analysis

of brain tissue.

Keywords: 2DE; Brain tissue, Hydrophobic proteins, Paper bridge; Cup Loading, HED.

1. Introduction

Proteomic investigation of normal and diseased brain states is able to reveal novel molecular

therapeutic and diagnostic targets for a multitude of pathological central nervous system

conditions. Changes of protein levels as well as modifications that occur in neurological

disorders may be informative for the pathogenesis of these disorders and could result in the

identification of potential drug targets and disease markers. 2-DE, the most popular tool to

explore neurodegenerative disorders, remains an efficient technique that allows not only a

screening for abundant-protein changes in various diseases, but also for alterations in

metabolic pathways. The major advantage of 2-DE lies in its potential to simultaneously

resolve thousands of proteins, at the same time revealing their MW, pI, and reflecting changes

in protein expression and isoforms[1]. Apart from these advantages, 2-DE has also some

drawbacks. One of its limitations includes the difficult identification of low (<15 kDa) and

high (>150 kDa) MW proteins and separation is generally limited to proteins that are neither

too acidic/basic, nor too hydrophobic. Nevertheless, the map is biased to proteins that are

present in larger amount or are hydrophilic, as compared to low-abundance and membrane-

spanning or less soluble proteins. Recently, several method improvements were suggested in

order to obtain an unbiased map by increasing the recovery of problematical proteins [2].

Sample solubility can be improved by using appropriate mixtures of chaotropic agents and

new efficient detergents [3, 4]. Replacing dithiothreitol with tributylphosphine or TCEP is

reported to enhance protein solubility during IEF, therefore increasing resolution and

recovery[5] . or using an alternative reducing agent such as hydroxyethyldisulphide (HED) to

form mixed disulfides with cysteinyl thiols to reduce the streaks caused by reoxidation of

disulphide bridges primarily due to the depletion of the reducing agent, such as DTT or its

isomer dithioerythritol (DTE) in the basic pH range during the first dimension IEF[6-8].

Moreover, literature data suggest that the alkylation of free thiolic groups should be

performed prior to the IEF to reduce spurious spots[9]. At the same time, it is very important

the way in which the sample is applied to an IPG strip and the amount of protein loaded. The

impact of different sample loading techniques on gel quality has received considerable

attention over the years. There are three different sample application methods commonly used

in 2-DE: active rehydration, passive rehydration, and cup-loading[10]. At present the most

commonly used micropreparative sample application method is to add sample to the solution

used for reswelling of the IPG drystrip (in-gel rehydratation)[11]. Compared to traditional cup

application in gel rehydratation allows the use of larger sample volumes but also generates

problems related to poor protein solubility, leading to loss of protein and streaking in the

focusing dimension particularly with membrane proteins and large proteins[4]. Many

investigators have demonstrated that qualitatively better separations can be obtained if the

sample is applied using the cup-loading method, but the application of a relatively large

quantity of protein (1 mg) in a small volume (about 100 μl) result in protein precipitation and

aggregation at the point of application with a general massive loss of proteins[12]. Another

method of sample loading called “paper-bridge loading” permit to increase the sample volume

(up to 2,5 ml) and therefore avoid streaking and protein loss by applying the sample to filter

papers placed as bridges between the end of IPG strip and the electrodes[12]. Our present

research involves rats as animal models for psychiatric and neurological disorders. The ideal

animal models should be similar to clinical cases in terms of etiology, biochemistry,

symptoms and treatment. The proteomic analysis of the brain has certain limitations that are

related either to the sample and/or analytical approach. The technical limitations involve

inefficient detection of low-abundance gene products, hydrophobic proteins (they do not enter

the IPG strips), and basic proteins . All these protein classes are under-expressed in brain 2-D

gels[13-16]. In this work we use a combination of proteomics methods and alternative

methodologies of rat brain protein sample extraction (Trizol vs Urea only), reduction (DTT vs

TCEP), alkylation (HED) and loading (Paper-bridge vs Cup-loading and passive

rehydratation) . Our goal was to evaluate different techniques for resolving the brain proteins

in a mixture, using IPG 3–10 strips. A analytical loading of 100 μg was used in all

experiments. The number of spots detected in each experiment, the reproducibility of each

sample loading technique, the gel-to-gel matching efficiency, and the reproducibility of spot

quantity with each technique was evaluated for the several independent preparations in order

to determine which technique is optimum and most reliable for separating the brain protein

components.

2. Materials and Methods

Rats were killed at 16 weeks of age, whole brains were rapidly removed, washed with PBS

with protease and phosphatase inhibitors, and brain areas of interest were cut on ice. Each

sample was immediately added with protease inhibitor cocktail (Sigma) and phosphatase

inhibitor cocktail (Sigma), flash frozen in liquid nitrogen, and stored at –80°C.

Protocol I: The tissue was weighed, suspended in solution of 7M urea, 2M thiourea, 2%

CHAPS, 2% Triton X-100, 15mM Tris, 1% ampholine 3.5-10 (1:10, w/v). The reducing

agents utilized in solution were 65mM DTT (dithiothreitol) or 10 mM TCEP [Tris (2-

carboxyethyl) phosphine hydrochloride]. The suspension was homogenized under mechanic

stirring for 3h. The supernatant was centrifuged at 10.000 xg for 30 min. The samples were

treated with HED solution (100mM) for 1h a RT. Protein concentrations were determined

using the 2D-Quant Kit (GE Healthcare). Samples were immediately loaded for the first

dimension.

Protocol II: The tissue was weighed and extraction of protein was perfomed with Trizol like

protocol according to the manufacturer's specifications (Omnizol - Euroclone). The proteins

were solubilized in 7M urea, 2M thiourea, 2% CHAPS, 2% Triton X-100, 15mM Tris, 1%

ampholine 3.5-10, (1:10, w/v). The reducing agents utilized in solution were 1% DTT or 10

mM TCEP. The samples were treated with HED (Hydroxyethyl disulfide) solution (100mM)

for 1h. Protein concentrations were determined using the 2D-Quant Kit (GE Healthcare).

Samples were immediately loaded for the first dimension.

Two-dimensional gel electrophoresis:

I Dimension (Isoelectric focusing - IEF): pH 3–10NL 18cm long IPG strips (GE Healthcare)

were used. The IPG strips were rehydrated overnight at room temperature using an

Immobiline DryStrip reswelling tray. The volume of rehydration solution was 350 μl for 18

cm strips and 100 μg of brain proteins were loaded with following methods: i) cup loading,

ii) paper bridge and iii) passive rehydration. IEF was then performed on a IPGPHOR 3

apparatus (GE Healthcare) at 20°C by a series of increasing voltage “steps” from 50 V to

8000 V, until the total voltage of 200.000 VhT was reached.

II Dimension (SDS-PAGE): After the first dimension, IPG strips were equilibrated twice for

15 min under gentle stirring with a solution containing 6 M urea, 2% SDS, 50 mM Tris-HCl

pH 8.8 and 30% glycerol. To the first equilibrium, 1% DTT was added and to the second

2.5% iodoacetamide and a trace of bromophenol blue. The second dimension was performed

by use homemade 10% acrylamide vertical SDS-PAGE slab gels of dimension

200x200x1mm. The samples were run overnight on a Ettan Dalt six electrophoresis unit (GE

Healthcare) at constant temperature of 20°C and limited to 1.0 W/gel. After the runs, gels

were stained with silver nitrate. Molecular masses are determined by analyzing standard

protein markers that cover the range 10–200 kDa. pI values are used as given by the supplier

of the IPG strips. The gels are scanned in a PHAROS FX apparatus (Bio-Rad). Electronic

images of the gels are recorded with Quantity One (Bio-Rad) software. The images were

stored in TIFF (about 5 Mbytes/file) and JPEG (about 50 Kbytes/file) format. Image analysis

was performed using ImageMaster 2D Platinum v6.0.1 software (GE Healthcare).

3. Results and Discussion

Appropriate sample preparation is essential for obtaining reliable results in a proteomic

analysis. Differences in the protein concentrations were observed between extraction

protocols (Fig 1).

Fig. 1 Schematic extraction protocols using buffer solution (I) and OMNIzol (II)

The recovered protein amount using protocol I was four time more than OMNIzol

precipitation (Fig. 2). In fact the protein precipitation usually results in protein losses and also

causes difficulties in re-solubilization of proteins.

Brain extraction protocols

extraction protocol

Buffer solution OMNIzol

mg

prot

ein/

ml b

uffe

r

0

2

4

6

8

10

Fig. 2 Extraction of total protein using protocol I and II (see materials and methods).

As show in figure 3, no significant differences were observed between extraction protocols.

Therefore the protocol I is the best choice for extraction of total protein because it allowed

better recovered protein amount and a rapid, simple, reproducible using for extraction protein

from brain tissue.

Fig. 3 Brain protein separation by SDS-PAGE (AA 8-16%) using two different protocols (I,

II) and two different reducing agents (DTT, TCEP).

Experiments carried out with various loading sample methods were shown different number

of detected spots. A total of 100 µg proteins were separated. About 948, 696, 587 spots were

detected on the 2-D gels of brain loaded by means of anodic cup loading, paper bridge and

passive rehydration, respectively (Fig. 4). The increase in the number of spots is most

probably due to different loading sample methods used.

Image Analysis

loading methods

cup loading paper bridge passive rehydration

tota

l spo

t num

ber

0

200

400

600

800

1000

Fig. 4 Number of detected spots using different loading methods

To by-pass the problem of streaking in the basic part of the strip during isoelectric focusing

was used HED. The use of HED was shown to remove much of the streaking found in the

basic pH region of 2-DE gels resulting in a more simplified spot pattern with improved

resolution and separation than samples without HED (Fig 5). Overall, this comparison has

determined the optimal methodology required for improved separation of brain proteins in

enlarged pH gradients in the IEF dimension. The paper bridge method showed the best results

in terms of spot resolution and separation of all proteins especially basic and high molecular

weight proteins (fig. 5). Unfortunately the number of spots detected in gels made with this

method was about 20% lower than those obtained with cup-loading procedure, this is

probably due to the various types of interactions between filter paper and sample. A

combination of using HED, when rehydrating the IPG strips, with cup-loading of the sample

at the anode prior to isoelectric focusing, showed the highest number of spots detected and

greatly improves the separation of all protein from rat brain tissue but especially basic

proteins. This method will improve the reproducibility of gels and the detection of additional

differences between diseased and control brains. Such differences may be essential in the

discovery of early disease markers and therapeutic approaches.

Fig 5. 2-D gel images of rat brain proteins. Separation was performed using different

extraction protocols I: (A) HED, (B) without HED, (C) HED, (D) without HED and different

loading methods: (A) (B) paper bridge, (C) (D) cup loading. Proteins were visualized by

staining with silver nitrate.

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