2. Material and methods

## 2.1. Materials

1. Introduction

126 Cholesterol - Good, Bad and the Heart

Smith-Lemli-Opitz syndrome (SLOS), OMIM #270400, is one of the nine known disorders associated with altered post-squalene cholesterol biosynthesis [1, 2]. This autosomal recessive genetic disease was first described in 1964, as a syndrome of cognitive impairment and multiple malformations [3]. Thirty years were needed to further characterize the disorder as a metabolic disease and identify the underlying enzymatic defect. SLOS is caused by deficiency of 7-dehydrocholesterol redutase (7-DHCR, 3-hydroxysteroid redutase, EC.1.3.1.21) which catalyzes the conversion of 7-dehydrocholesterol (7DHC) to cholesterol, the terminal step of Kandutsch-Russell pathway [4, 5]. Consequently, SLOS patients typically show increased 7DHC and decreased serum and tissue cholesterol levels [5]. In 1998, mutations of the 3βhydroxysterol Δ7-reductase gene (DHCR7) were shown to cause SLOS [6–8] and more than 154 DHCR7 mutations have been so far identified in SLOS patients [9]. The development of animal models has improved the understanding of SLOS physiopathology and provided material for in vivo and in vitro investigation of biological consequences of cholesterol deficiency. In 2001, two mouse models with null mutations in Dhcr7 gene have been created by homologous recombination [10, 11]. The malformations in the null mice are very mild compared to what would be seen in a null human infant (i.e., SLO type II). The mutant mice died within the first 24 h of extra-uterine life. Later, a mouse model with a milder phenotype was developed [12]. This hypomorphic mouse has a missense mutation equivalent to the human p.T93M, previously identified in SLOS patients often with Mediterranean heritage [13–15]. Like the majority of SLOS patients, the SLOS mouse models manifest a deficiency in choles-

terol biosynthesis resulting in low levels of cholesterol in serum and tissues [11].

with some of them being involved in the crosstalk between organelles [23].

Effective cholesterol biosynthesis is especially critical at certain stages during development and continues to be important throughout life [16], since cholesterol is an essential lipid with multiple functions. Cholesterol is a major lipid component of membrane microdomains, which are crucial cell-surface dynamic structures responsible for many cellular signaling and communication events [17]. Membrane domains can form through a number of mechanisms involving lipid-lipid and protein-lipid interactions. One type of membrane domain is the cholesterol-dependent membrane raft [18]. Properties of these membrane domains have been primarily inferred from the study of detergent-resistant membranes (DRMs), composed by the non-ionic-detergent insoluble, low-buoyant density membranous fractions of cells [19]. Although it was initially thought that such microdomains enriched in cholesterol exist exclusively in the plasma membrane, increasing evidence suggests that similar lipid microdomains (sometimes referred as raft-like microdomains) are also present in internal organelles [20–22]

Proteomics constitutes a powerful tool to study complex biological mechanisms and to identify alterations in protein expression induced by changes in the environment, drugs, or disease states. As such, proteomics is now widely employed to help understand pathological processes induced by the disease [24–27]. The effect of an inborn error of cholesterol synthesis on skeletal muscle has not previously been reported. In this chapter, we report a comparative analysis of protein expression in skeletal muscle DRMs isolated from Dhcr7 T93M homozygous mutant Butylhydroxytoluene (BHT), N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA), triethylammonium bicarbonate (TEAB), trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (α-CHCA), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), protease inhibitor cocktail, formic acid and urea were purchased from Sigma (Karlsruhe, Germany). RC DC Protein Assay kit for protein quantification was from BioRad lab (Hercules, USA). The iTRAQ kit was purchased from Applied Biosystems (Foster City, CA). Sequencing grade modified trypsin (bovine) was from ABSciex (ABSciex, USA). HPLC-grade acetonitrile (ACN, Riedel, Seelze, Germany) and Milli-Q grade water were also used. Rabbit raised polyclonal anti-caveolin 1 (ab2910) and anti-annexin A2 antibodies were purchased from Abcom, Cambridge, UK. Analytical reagent grade chemicals were used unless stated otherwise.

#### 2.2. Animals

The T93M mutation [12] was backcrossed into C57/BL6 for three generations. Homozygous (T93M/T93M) mice are viable and fertile [16]. Control C57/BL6 mice were obtained from IBMC Animal Centre from Oporto University.

All the animals were housed in plastic cages with free access to water and food (cholesterol free —chow -Mucedola, Ref: 4RF21). The animals were handled and maintained in controlled conditions according to international standards. Animals were euthanized using deep isoflurane anesthesia when they were 10 days old. Gastrocnemius and soleus muscles were dissected, submerged in ice-cold buffer (TRIS, HCl, pH 7.4, 10 mM mercaptoethanol, 0.28 M sucrose) and immediately frozen at 80C.

#### 2.3. DRMs extraction

Detergent-resistant membranes (DRMs) were isolated using cold Triton X-100 treatment followed by sucrose gradient centrifugation. In order to minimize individual variation, samples were analyzed as pools of several animals. The procedure was adapted from Kim et al. [28]. Briefly, the samples were allowed to thaw slowly on ice, and tissue from three mice (approximately 300 mg) was minced with scissors, mixed with 700 µL of cold (4C) lysis buffer (25 nM HEPES-HCl, pH 6,5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitor cocktail) and homogenized 30 times with a Poter homogenizer. The sample was maintained in an ice bath during homogenization and then incubated for 30 min at 4C. Aliquots of this homogenate were collected for protein quantification and sterol analysis.

2.5. Proteomic profile

the calibration curve.

raphy as previously published [30].

from the PANTHER classification system.

2.6. Immunocytochemistry assay

2.5.3. LC-MS/MS analysis

2.5.1. Pellet solubilization and protein quantification

2.5.2. Sample preparation for mass spectrometry analysis

30 min at 4C and saved for analysis. Insoluble material was discarded.

Each assay involved four sample pools: a wt-BL6 control and a Dhcr7T93M/T93M and their duplicates. For protein extraction, the DRMs pellets were treated with 10 volumes (w/v) of solubilization buffer (7 M urea, 2 M thiourea, 1% (w/v) CHAPS, 1% (w/v) Triton X-100, 1% (v/v) ampholytes (3–10) and 1 mM TCEP), sonicated briefly, then incubated for 10 min at room temperature under agitation. Clear cell lysates were obtained by centrifugation at 12,000g for

Quantitative Proteomic Analysis of Skeletal Muscle Detergent-Resistant Membranes in a Smith-Lemli-Opitz…

http://dx.doi.org/10.5772/intechopen.78037

129

The protein concentration was determined by RC DC Protein Assay kit, using BSA to generate

Reduction, alkylation and digestion steps were performed according to the protocol provided by the manufacturer. Briefly, 100 mg of DRM protein was mixed with TEAB buffer (1 M, pH 8.5) and an enhancer of enzymatic digestion RapiGest (Waters) to give a final concentration of 0.5 M and 0.1%, respectively. Samples were then treated with: (1) a reducing agent—TCEP 5 mM—used to break disulfide bonds within and between proteins, for 1 h at 37C and (2) a cysteine (sulfhydryl group) blocking reagent—S-methyl methanethiosulfonate (MMTS) 10 mM for 10 min, at room temperature. Then, 2 mg of trypsin was added to each sample and the digestion was performed for 18 h at 37C. The digested tryptic specimens were dried using a Speed-Vac. iTRAQ labeling was carried out according to the instructions provided by the manufacturer. Dhcr7T93M/T93M mice DRMs samples were marked with iTRAQ Tags 116 and wt-BL6 with114 and the duplicates with 115 and 113, respectively. The labeled samples were combined in pairs and dried in a Speed-Vac. The peptides were separated by reverse-phase liquid chromatog-

Peptide mass spectra were obtained in the mass range 700–4500 Da on a MALDI-TOF/TOF mass spectrometer (4800 Proteomics Analyzer, Applied Biosystems, Foster City, CA, USA) in the positive ion reflector mode. The obtained spectra were processed and analyzed by the ProteinPilot® software (v4.0 AB Sciex, USA), which uses an algorithm for protein/peptide identification based on

Protein clustering was performed according to biological and molecular functions derived

Immunocytochemistry assays were performed in order to confirm the presence of (1) caveolin-1, a protein marker of a subtype of membrane microdomains which are rich in cholesterol, designated caveolae (anti-caveolin-1 antibody dilution 1:500) and (2) annexin 2 another protein associated with enriched cholesterol microdomains, that was found increased in the SLO

the comparison of MS/MS data against the SwissProt protein database [31].

The resulting extract was mixed with an equal volume of cold sucrose 80% (w/v) to give a final sucrose concentration of 40%, transferred to the bottom of a ultra-centrifuge tube (Ultra-clear 14 89 mm, Beckman Ref 344059) and overlaid carefully with 6.0 mL 30% and 3.0 mL 5% cold sucrose solutions, containing 25 nM HEPES-HCl, pH 6.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100. Discontinuous sucrose gradients were centrifuged for 20 h, at 187,000 g on a Sorvall Ultra Pro 80 UC, swinging bucket SW41 at 4C. The DRMs fraction, which collects at the interface of the 5 and 30% sucrose layers, was isolated, washed with 9 mL of modified HEPES buffer and centrifuged 30 min at 49,500 g at 4C. After centrifugation, the supernatant was discarded and the pellet containing purified DRMs was suspended in phosphate buffered saline (PBS) and stored frozen at 80C until further proteomic or sterol analysis.

#### 2.4. Sterols analysis

For extraction of neutral sterols, 50 µL of sample was mixed with 50 µL of BHT 0.05% in methanol (antioxidant), 50 µL of epicoprostanol 0.10 mmol/L (internal standard) and saponified at 60C for 1 h by 16 adding 1 mL of 4% (w/v) KOH in 90% ethanol. The samples were then diluted with 1 mL of deionized water and the lipids were extracted twice with 2 mL of hexane. The pooled hexane extracts were dried under a gentle nitrogen stream at room temperature and derivatized with 50 µL BSTFA in 50 µL pyridine at 60C for 1 h [29]. The trimethylsilylether derivatives of sterols were separated by GC–MS with a Supelco 28471\_U SLB-5MS column (30 m 0.32 mm i.d., 0.25 μm film thickness). The injector temperature was set at 270C, and the splitless injection mode was used. The initial column temperature was 180C for 1 min and programmed to increase at a rate of 20C/min to 250C and then increased again at 5C/min rate till 300C. The carrier gas was helium at a linear constant flow rate of 40 cm/s. The interface was programmed to 280C, the quadrupole to 150C and the ionization source to 230C. After a solvent delay of 2.5 min, the eluted sterols were identified by their retention times (comparing with commercial standards) and respective mass spectra and quantified by selected ion monitoring (Table 1). The method was linear and reproducible for the range of amounts assayed.


The m/z ions selected for sterol quantification are printed in bold.

Table 1. Sterol parameters.

### 2.5. Proteomic profile

an ice bath during homogenization and then incubated for 30 min at 4C. Aliquots of this

The resulting extract was mixed with an equal volume of cold sucrose 80% (w/v) to give a final sucrose concentration of 40%, transferred to the bottom of a ultra-centrifuge tube (Ultra-clear 14 89 mm, Beckman Ref 344059) and overlaid carefully with 6.0 mL 30% and 3.0 mL 5% cold sucrose solutions, containing 25 nM HEPES-HCl, pH 6.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100. Discontinuous sucrose gradients were centrifuged for 20 h, at 187,000 g on a Sorvall Ultra Pro 80 UC, swinging bucket SW41 at 4C. The DRMs fraction, which collects at the interface of the 5 and 30% sucrose layers, was isolated, washed with 9 mL of modified HEPES buffer and centrifuged 30 min at 49,500 g at 4C. After centrifugation, the supernatant was discarded and the pellet containing purified DRMs was suspended in phosphate buffered

For extraction of neutral sterols, 50 µL of sample was mixed with 50 µL of BHT 0.05% in methanol (antioxidant), 50 µL of epicoprostanol 0.10 mmol/L (internal standard) and saponified at 60C for 1 h by 16 adding 1 mL of 4% (w/v) KOH in 90% ethanol. The samples were then diluted with 1 mL of deionized water and the lipids were extracted twice with 2 mL of hexane. The pooled hexane extracts were dried under a gentle nitrogen stream at room temperature and derivatized with 50 µL BSTFA in 50 µL pyridine at 60C for 1 h [29]. The trimethylsilylether derivatives of sterols were separated by GC–MS with a Supelco 28471\_U SLB-5MS column (30 m 0.32 mm i.d., 0.25 μm film thickness). The injector temperature was set at 270C, and the splitless injection mode was used. The initial column temperature was 180C for 1 min and programmed to increase at a rate of 20C/min to 250C and then increased again at 5C/min rate till 300C. The carrier gas was helium at a linear constant flow rate of 40 cm/s. The interface was programmed to 280C, the quadrupole to 150C and the ionization source to 230C. After a solvent delay of 2.5 min, the eluted sterols were identified by their retention times (comparing with commercial standards) and respective mass spectra and quantified by selected ion monitoring (Table 1). The method was linear and reproducible for

homogenate were collected for protein quantification and sterol analysis.

saline (PBS) and stored frozen at 80C until further proteomic or sterol analysis.

Sterol Retention time m/z

Desmosterol 14.10 343 327

The m/z ions selected for sterol quantification are printed in bold.

Epicoprostanol 13.02 370 257 355 Cholesterol 13.72 329 353 368 7DHC 14.02 325 351 366

Sitosterol 14.80 357 396 486 Lathosterol 15.80 255 443 458

2.4. Sterols analysis

128 Cholesterol - Good, Bad and the Heart

the range of amounts assayed.

Table 1. Sterol parameters.

## 2.5.1. Pellet solubilization and protein quantification

Each assay involved four sample pools: a wt-BL6 control and a Dhcr7T93M/T93M and their duplicates. For protein extraction, the DRMs pellets were treated with 10 volumes (w/v) of solubilization buffer (7 M urea, 2 M thiourea, 1% (w/v) CHAPS, 1% (w/v) Triton X-100, 1% (v/v) ampholytes (3–10) and 1 mM TCEP), sonicated briefly, then incubated for 10 min at room temperature under agitation. Clear cell lysates were obtained by centrifugation at 12,000g for 30 min at 4C and saved for analysis. Insoluble material was discarded.

The protein concentration was determined by RC DC Protein Assay kit, using BSA to generate the calibration curve.
