Pi 87782

MLRs protein samples were diluted in TNE (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA) buffer. RapiGest SF reagent (Waters) was added to the mix (0.1% final concentration) and samples boiled for 5 min. 1 mM TCEP (Tris [2-carboxyethyl] phosphine) was added to the samples and incubated at 37°C for 30 min. Samples were carboxymethylated with 0.5 mg/mL of iodoacetamide for 30 min at 37°C followed by neutralization with 2 mM TCEP. Samples were digested with trypsin (trypsin: protein ratio 1:50) overnight at 37°C. RapiGest was degraded and removed by treating the samples with 250 mM HCl at 37°C for 1 h followed by centrifugation at 14k rpm for 30 min at 4°C. The soluble fraction was then added to a new tube and the peptides were extracted and desalted using C18 desalting columns (Thermo Scientific, PI-87782). Peptides were quantified using BCA assay and a total of 1 μg of peptides were injected for liquid chromatography-MS (LC-MS) analysis.

Protein samples were diluted in TNE (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA) buffer. RapiGest SF reagent (Waters Corp.) was added to the mix to a final concentration of 0.1% and samples were boiled for 5 min. TCEP (Tris (2-carboxyethyl) phosphine) was added to 1 mM (final concentration) and the samples were incubated at 37° for 30 min. Subsequently, the samples were carboxymethylated with 0.5 mg/ml of iodoacetamide for 30 min at 37° followed by neutralization with 2 mM TCEP (final concentration). Protein samples prepared as above were digested with trypsin (trypsin:protein ratio - 1:50) overnight at 37°. RapiGest was degraded and removed by treating the samples with 250 mM HCl at 37° for 1 h followed by centrifugation at 14000 rpm for 30 min at 4°. The soluble fraction was then added to a new tube and the peptides were extracted and desalted using C18 desalting columns (Thermo Scientific, PI-87782)(Guttman et al., 2009 (link); McCormack et al., 1997 (link); Shevchenko et al., 1996 (link)).