# Muscular and Molecular Pathology Associated with SPATA5 Deficiency in a Child with EHLMRS
## 4 Materials and Methods
### Human Muscle Preparation for Mass Spectrometry  
Surgically removed muscle tissue was snap-frozen in liquid nitrogen and stored at \(-\)80 \({}^{\circ}\)C. In addition to the patient-derived muscle sample (measured in technical triplicates), muscle biopsies of three gender and age-matched individuals were included and defined as non-diseased controls (NDCs). Here, we selected \"patients\" that had undergone a muscle biopsy due to nonspecific complaints but were found not to have any inflammatory muscle disease, e.g., they suffered from myalgia, but objective muscle weakness and morphological abnormalities on the skeletal muscle biopsy were absent. CK levels were normal and no signs of systemic inflammation, myositis-specific antibodies, (MSA) or myositis-associated antibodies (MAA) were detectable. The total muscle was lysed in 200 \(\upmu\)L of 50 mM Tris-HCl (pH 7.8) buffer, 5% SDS, and cComplete ULTRA protease inhibitor (Roche) using the Bioruptor(r) (Diagenode) for 10 min (30 s on, 30 s off, 10 cycles) at 4 \({}^{\circ}\)C. To ensure complete lysis, an additional sonication step using an ultra-sonic probe (30 s, 1 s/1 s, amplitude 40%) followed by centrifugation at 4 \({}^{\circ}\)C and 20,000\(\times\)_g_ for 15 min was completed. Protein concentration of the supernatant was determined by a BCA assay according to the manufacturer's protocol. Disulfide bonds were reduced by the addition of 10 mM TCEP at 37 \({}^{\circ}\)C for 30 min, and free sulfhydryl bonds were alkylated with 15 mM IAA at room temperature (RT) in the dark for 30 min. In each sample, 100 \(\upmu\)g protein was used for proteolysis using the S-Trap protocol (Protifi) and using a protein to the trypsin  
\begin{table}
\begin{tabular}{c c c c} \hline
**Primary Antibody, Clone** & **Company** & **Number** & **Dilution** \\ \hline Caveolin 3, 26/Caveolin3 & BD Transduction Laboratories & 610420 & 1:500 \\ Dystrophin 1, Dy4/6d3 & Novocastra/Leica & NCL-DY51 & 1:2 \\ Dystrophin 2, Dy8/6c & Novocastra/Leica & NCL-DY52 & 1:10 \\ Dystrophin 3, Dy1/12B2 & Novocastra/Leica & NCL-DY53 & 1:10 \\ \(\upalpha\)-Dystroglycan, VIA4-1 & Merck/Upstate & \#05-298 & 1:15 \\ \(\upbeta\)-Dystroglycan, 43 DAG1/8D5 & Novocastra/Leica & NCL-b-DG & 1:10 \\ \(\upalpha\)-Sarcoglycan, Ad1/20A6 & Novocastra/Leica & NCL-a-SARC & 1:50 \\ \(\upbeta\)-Sarcoglycan, 9 Sarc/5B1 & Novocastra/Leica & NCL-b-SARC & 1:50 \\ \(\upgamma\)-Sarcoglycan, 35 DAG/21B5 & Novocastra/Leica & NCL-g-SARC & 1:50 \\ \(\updelta\)-Sarcoglycan, 35 DAG/21B5 & Novocastra/Leica & NCL-d-SARC & 1:10 \\ Laminina \(\upalpha\)C-term, 5H2 & Merck/Chemicon & MAB1922 & 1:500 \\ Emera, 4G5 & Novocastra/Leica & NCL-EMERIN & 1:20 \\ Dysferlin, Hamlet 1 & Novocastra/Leica & NCL-HAMLET & 1:5 \\ Collagen 6, VI-26 & Merck/Chemicon & MAB3303 & 1:100 \\ Collagen 6, 3C4 & Merck/Chemicon & MAB1944 & 1:200 \\ Collagen 4, polyclonal & Cedarline & CL50411 AP & 1:100 \\ Laminina \(\upalpha\)5, 4C7 & Merck/Chemicon & MAB1924 & 1:500 \\ DRP 2 (Utrophin), DRP3/20C5 & Novocastra/Leica & NCL-DRP2 & 1:5 \\ Neonatal Myosin, WB-MHCh & Novocastra/Leica & NCL-MHCn & 1:5 \\ \hline \end{tabular}
\end{table}
Table 3: Primary antibodies used in immunohistochemistry.  
ratio of 20:1. The incubation time for trypsin was changed to 2 h at 37 \({}^{\circ}\)C. Proteolysis was stopped using TFA to acidify the sample (pH < 3.0).  
All proteolytic digests were checked for complete digestion after desalting by using monolithic column separation (PepSwift monolithic PS-DVB PL-CAP200-PM, Dionex, Germering, Germany) on an inert Ultimate 3000 HPLC (Dionex, Germering, Germany) by direct injection of a 1 \(\upmu\)g sample. A binary gradient (solvent A: 0.1% TFA, solvent B: 0.08% TFA, 84% ACN) ranging from 5-12% B in 5 min and then from 12-50% B in 15 min at a flow rate of 2.2 \(\upmu\)L/min and at 60\({}^{\circ}\), was applied. UV traces were acquired at 214 nm [23].