Nanolc ultra 2d nano hplc system

Two hundred μL of m-Aminophenylboronic acid-Agarose beads (Sigma) were washed 3 times with 1 mL of binding buffer (200 mM NH4Ac, 30 mM MgCl2, pH 8,9) and incubated with 1 mL of NMN (Oriental Yeast Co., Ltd.) or CMP (Sigma) solution (5 g/l in binding buffer) for 1 hour at room temperature. Remaining concentration of NMN and CMP in the solution were monitored using NanoDropONE spectrophotometer (Thermo Fisher) by absorbance at λ = 270 nm. Beads were washed 3 times with binding buffer and incubated with 1 mL of 2:1 mixture of binding buffer and solubilized membrane proteins purified from glioblastoma cells. After overnight incubation at +4 o C with constant agitation, beads were washed once with 2:1 mixture of binding buffer and solubilization buffer (Thermo Fisher) and 4 times with binding buffer. After the last wash NMN, CMP and bound proteins were eluted with 100 μL of 25 mM HCl. Immediately after elution, the eluate was neutralized with 1 M Tris buffer pH 9. Eluted proteins were subjected to LC-MS/MS analysis, performed on a TripleTOF 5600+ mass-spectrometer with a NanoSpray III ion source (ABSciex) coupled with a NanoLC Ultra 2D+ nano-HPLC system (Eksigent) as described previously (35) . Proteins eluted from empty beads (without NMN or CMP) were used as a negative control.