Neuronal tissue was separated using the XCell SureLock Mini-Cell system (Thermo, EI0001). Gel was stained using SimplyBlue (Invitrogen, LC6060). Band excised at 32.5 kDa was denature using 6 M urea, reduced in 10 mM Dithiothreitol, alkylated in 15 mM Iodoacetamide, and quenched in 20 mM Dithiothreitol. Samples were digested with either Trypsin (Promega, V511A), ChymoTrypsin (Promega, V106A), or Proteinase K (Thermo, 17916). Mass spectrometry was performed on a Q-Exactive instrument after fractionation on a coupled Easy nLC 1000 nano-liquid chromatography system (Thermo Fisher Scientific, Waltham, MA) as described in our other published reports [22 (link)].
Protocol detail
Neuronal Protein Identification Workflow
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Cell
Chymotrypsin
Dithiothreitol
Fractionation
Iodoacetamide
Liquid chromatography
Mass spectrometry
Neuronal
Promega
Proteinase k
Tissue
Trypsin
Urea
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Variable analysis
independent variables
- Protein digestion enzyme (Trypsin, Chymotrypsin, or Proteinase K)
- Protein identification and characterization by mass spectrometry
- Neuronal tissue separation using the XCell SureLock Mini-Cell system
- Gel staining using SimplyBlue
- Protein denaturation using 6 M urea
- Protein reduction using 10 mM Dithiothreitol
- Protein alkylation using 15 mM Iodoacetamide
- Quenching of alkylation using 20 mM Dithiothreetol
- Nano-liquid chromatography system (Easy nLC 1000)
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