C18 zip tip solid phase extraction

Anterior horns of the spinal cord samples were processed for protein extraction. Frozen Neurological post-mortem tissue samples were collected and homogenized in lysis buffer containing 7 M urea, 2 M thiourea and 50 mM DTT by mechanical disruption assisted by a Potter (Sartorius, Potter S, Goettingen, Germany). The resulting homogenates were ultracentrifuged at 100,000× g for 1 h at 15 °C. Prior to proteomic analysis, protein extracts were precipitated with methanol/chloroform, and pellets dissolved in 6 M Urea, Tris 100 mM pH 7.8. Protein quantitation was performed with the Bradford assay kit (Bio-Rad, Hercules, CA, USA) and 100 µg of each protein extract were subjected to enzymatic digestion using trypsin (Promega; ratio 1:50, w/w) at 37 °C for 16 h. Purification and concentration of peptides was performed using C18 Zip Tip Solid Phase Extraction (Millipore, Burlington, MA, USA).

Whole OB specimens (70–80 mg) derived from controls and ALS cases were homogenized using ‘’mini potters’’ in lysis buffer containing 7 M urea, 2 M thiourea and 50 mM DTT. The homogenates were spun down at 100,000× g for 1 h at 15 °C. Before proteomic analysis, protein extracts were precipitated and pellets were dissolved in 6 M Urea and Tris 100 mM pH 7.8. Protein quantitation was performed with the Bradford assay kit (Bio-Rad). The protein extract for each sample was diluted in Laemmli sample buffer and loaded into a 0.75-mm-thick polyacrylamide gel with a 4% stacking gel casted over a 12.5% resolving gel. The run was stopped as soon as the front entered 3 mm into the resolving gel so that the whole proteome became concentrated in the stacking/resolving gel interface. Bands were stained with Coomassie Brilliant Blue and excised from the gel. Protein enzymatic cleavage (10 ug) was carried out with trypsin (Promega; 1:20, w/w) at 37 °C for 16 h as previously described (Shevchenko et al., 2006). Peptides were purified and concentrated using C18 Zip Tip Solid Phase Extraction (Millipore, Burlington, MA, USA).

Nuclear extracts from Mock-infected, and U87-infected cells (at 6 and 10hpi) were diluted in Laemmli sample buffer and loaded into a 1 mm thick polyacrylamide gel with a 4% stacking gel casted over a 12.5% resolving gel. The run was stopped as soon as the front entered 3 mm into the resolving gel so that the whole proteome became concentrated in the stacking/resolving gel interface. Bands were stained with Coomassie Brilliant Blue and excised from the gel. Protein enzymatic cleavage (15ug) was carried out with trypsin (Promega; 1:20, w/w) at 37°C for 16 h as previously described [59 (link)]. Purification and concentration of peptides was performed using C18 Zip Tip Solid Phase Extraction (Millipore).

Eluates were homogenised in lysis buffer (7 M urea, 2 M thiourea, 50 mM DTT) and the protein concentration was quantified with the Bradford assay (Bio-Rad, Hercules, CA, USA) and then precipitated with a ReadyPrep 2-D cleanup kit (Bio-Rad). The protein extract for each sample was diluted in Laemmli buffer and loaded into a 1.5 mm thick polyacrylamide gel with a 4% stacking gel cast over a 15% resolving gel. The run was stopped as soon as the front entered 3 mm into the resolving gel to concentrate the whole proteome in the stacking/resolving gel interface. Bands were stained with Coomassie Brilliant Blue and excised from the gel. Protein enzymatic cleavage was carried out with trypsin (Promega, Madison, WI, USA; 1:20, w/w) at 37 °C for 16 h, as previously described [22 (link)]. Purification and concentration of peptides was performed using C18 Zip Tip Solid Phase Extraction (Millipore, Burlington, MA, USA).