2.5.2. Sample preparation for mass spectrometry analysis

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 chromatography as previously published [30].

#### 2.5.3. LC-MS/MS analysis

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

Protein clustering was performed according to biological and molecular functions derived from the PANTHER classification system.

#### 2.6. Immunocytochemistry assay

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 mouse model (antibody dilution 1:2000). A sequential incubation with a secondary biotinylated anti-rabbit antibody was performed and diaminobenzidine (which stains brown) was employed as chromogen. Skeletal muscle samples were then counterstained with hematoxylin.

3.2. Proteomics

In order to explore the protein changes on sarcolemma, due to decreased 7-DHCR activity, we

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

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

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A total of 133 unique proteins were identified. Those identified based on a single peptide and those with a protein score less than 2.5 fold were excluded. Then a cut-off of 30% was applied to iTRAQ average ratios allowing us to select proteins with an important variation in SLOS mice relatively to controls. Differential protein expression was specific and not just a general finding. Caveolin-1, a protein known to be expressed in cholesterol-rich membrane microdomains did

Of the 133 identified proteins, we observed an altered expression of 38 (29%) proteins. Increased and decreased expression was observed for 17 and 21 proteins, respectively (Table 2). The replicate samples demonstrated a strong positive correlation (r = 0.90) and indicated good

Most proteins showing an altered expression in DRMs preparations were found to participate in at least one of three main cellular processes: membrane trafficking, energy production and

Figure 2. Comparative analysis of sterol composition of DRMs extracted from skeletal muscle of wt-BL6 controls and

Figure 3. Immunohistochemical stain for caveolin-1 (10) shows no significant differences between Dhcr7 93M/93M (on the

analyzed DRMs utilizing iTRAQ labeling and LC-MS/MS.

not show differential expression (Figure 3).

reproducibility (Figure 4).

hypomorphic DHCR7T93M/T93M (SLOS) mice.

left) and wt-BL6 (on the right) skeletal muscle samples.

Ca2+ homeostasis.
