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Proteome profiling of human myoblastic RCMH cell line
The study aimed to comprehensively characterize human myoblastic cell line RCMH using using electron microscopic and proteomic approaches. Myoblastic cell lines can be useful to investigate the complex biochemical changes occuring under different conditions that reflect the physiological and pathophysiological mechanisms of muscle. So far, there are no suitable in vitro models of human muscle origin to study a variety of muscle related processes including responses to mechanical stress, EC-coupling and (ER-associated) myopathic disorders. Therefore, we characterized the human immortal myoblastic cell line RCMH and the results suggest RCMH as a suitable in vitro model for investigating human muscle related processes and disorders.
Sample Processing Protocol
Approximately 2 mg of cells were lysed using 0.5 mL of 50 mM Tris-HCl (pH 7.8) buffer containing 150 mM NaCl, 1% SDS and complete mini-EDTA free. Subsequently, 6 µL of benzonase (25 U/µL) and 2 mM MgCl2 were added to the lysate and incubated at 37°C for 30 min. The cell lysate was clarified by centrifugation at 20,000 rcf and 4°C for 30 min. To the supernatant, ice cold ethanol was added in 10-fold excess and stored at -40°C for 1 h followed by centrifugation as above. The pellet was washed with 200 µL of ice cold acetone and was allowed to dry under laminar flow hood. The protein pellet was re-solubilized with 0.3 mL of 6 M GuHCL/50 mM NH4HCO3 buffer (pH 7.8) and protein concentration was determined by BCA assay according to the manufacturer’s instructions (Pierce BCA Protein Assay Kit, Thermo Scientific). Cysteines were reduced with 10 mM DTT at 56°C for 30min and the free thiols were carbamidomethylated with 30 mM IAA at RT for 30 min in the dark. Sample cleaning and enzymatic proteolysis was carried out based on filter aided sample preparation (FASP) protocol. Finally, the peptides were acidified to ~pH 3 using 10% TFA (v/v). Acidified peptides were desalted with C18 solid phase extraction cartridges (SPEC, 4 mg, Varian) according to the manufacturer’s instructions and the eluted peptides were dried under vacuum. The dried peptide pellet was re-suspended in buffer A (10 mM ammonium acetate, pH 6.0) and an aliquot equivalent to 25 µg was fractionated on an Ultimate 3000 LC system (Thermo Scientific). Peptides were separated on a 1 mm x 150 mm C18 (ZORBAX 300SB) column with a 75 min LC gradient ranging from 3-50% buffer B (84% ACN in 10 mM ammonium acetate, pH 6.0) at a flow rate of 12.5 µL/min. In total, 16fractions were collected at 1 min intervals in concatenated manner. Each fraction was dried under vacuum and re-suspended in 15 µL of 0.1% TFA for nano-LC-MS/MS analysis. All 16 fractions were analyzed using an Ultimate 3000 nano RSLC system coupled to an Orbitrap Elite mass spectrometer (both Thermo Scientific). Peptides were preconcentrated on a 75 µm x 2 cm C18 trapping column for 10 min using 0.1% TFA (v/v) with a flow rate of 20 µL/min, followed by separation on a 75 µm x 50 cm C18 main column (both Pepmap, Thermo Scientific) with a 128 min LC gradient ranging from 3-42% B (84% ACN in 0.1% FA) at a flow rate of 250 nL/min. MS survey scans were acquired in the Orbitrap from 300 to 1500 m/z at a resolution of 60,000 using the polysiloxane ion at 371.101236 m/z as lock mass. The fifteen most intense ions were subjected to collision induced dissociation (CID) in the ion trap, taking into account a dynamic exclusion of 30 s. CID spectra were acquired with a normalized collision energy of 35% and an activation time of 10 ms. AGC target values were set to 106 for Orbitrap MS and 104 for ion trap MSn scans.
Data Processing Protocol
MS raw data were converted into peak lists (mgf files) using the ProteoWizard software (version 2.2.2954) and were searched against a concatenated target/decoy version of the human Uniprot database, (downloaded on 11th of December 2013, containing 20,273 target sequences) using Mascot 2.4 (Matrix Science), MS-GF+, and X!Tandem Jackhammer (2013.06.15) with the help of searchGUI 1.18.4. Trypsin with a maximum of two missed cleavages was selected as enzyme. Carbamidomethylation of Cys was set as fixed and oxidation of Met was selected as variable modification. MS and MS/MS tolerances were set to 10 ppm and 0.5 Da, respectively. The results of the different search algorithms were imported into the PeptideShaker software 0.29.1 (http://code.google.com/p/peptide-shaker/) for interpretation and validation of the spectrum, peptide and protein identifications using a false discovery rate (FDR) of 1% on the peptide and protein level. The quality controlled data was exported and only proteins that were identified with ≥ 1 validated peptide were considered for the data analysis. To roughly estimate the abundance of the identified proteins, NSAF (normalized spectral abundance factor) values were calculated and additional information like, subcellular location and disease was retrieved from the Uniprot website (http://www.uniprot.org/).
Laxmikanth Kollipara, Leibniz – Institut für Analytische
Wissenschaften - ISAS - e.V.
René P. Zahedi, Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V. Otto-Hahn-Str. 6b D-44227 Dortmund Germany Tel.: +49 (0) 231-1392-4143 Fax: +49 (0) 231-1392-4850 ( lab head )
Kollipara L, Buchkremer S, Weis J, Brauers E, Hoss M, Rütten S, Caviedes P, Zahedi RP, Roos A. Proteome profiling and ultrastructural characterization of the human RCMH cell line: myoblastic properties and suitability for myopathological studies. J Proteome Res. 2016 Jan 19 PubMed: 26781476
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||47551||no assay title provided (mzIdentML)||21070||53498||43817||50989||50037||
|2||47552||no assay title provided (mzIdentML)||21255||53886||44016||51601||50299||
|3||47550||no assay title provided (mzIdentML)||17885||38938||32986||37225||36396||
|4||47547||no assay title provided (mzIdentML)||18103||39545||33231||37774||36957||
|5||47546||no assay title provided (mzIdentML)||17816||38243||32029||36519||35762||
|6||47558||no assay title provided (mzIdentML)||18450||40436||34060||38493||37820||
|7||47549||no assay title provided (mzIdentML)||18385||40338||33845||38247||37727||
|8||47557||no assay title provided (mzIdentML)||18273||39951||33660||38062||37446||
|9||47548||no assay title provided (mzIdentML)||18505||40420||33996||38253||37681||
|10||47556||no assay title provided (mzIdentML)||18237||39600||33416||37755||37123||