Autoflex 3 smartbeam maldi tof tof

Protein extracts were separated by two-dimensional gel electrophoresis as previously described [53 (link)]. Gels were then labeled with Flamingo Fluorescent Gel Stain (Bio-Rad, Hercules, CA, USA), scanned in a Typhoon 9400 (GE Healthcare, Chicago, IL, USA), and analyzed for spot variations using PD-Quest 8.0 (Bio-Rad, Hercules, CA, USA). Three animals from each group were analyzed. Spots of interest were then excised and identified by matrix-assisted laser desorption/ionization-time-of-flight using an AutoFlex III Smartbeam MALDI-ToF/ToF (Bruker Daltonics, Billerica, MA, USA).

As previously described [55 (link)], proteins were identified after in-gel tryptic digestion and extraction of peptides from the gel’s pieces, by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) using an AutoFlex III Smartbeam MALDI-TOF/TOF (Bruker Daltonics, Barcelona, Spain). Samples were applied to Prespotted AnchorChip plates (Bruker Daltonics, Barcelona, Spain) surrounding calibrates provided on the plates. Spectra were acquired with flexControl on reflector mode, (mass range 850–4000 m/z, reflector 1: 21.06 kV; reflector 2: 9.77 kV; ion source 1 voltage: 19 kV; ion source 2: 16.5 kV; detection gain 2.37×) with an average of 3500 added shots at a frequency of 200 Hz. Each sample was processed with flexAnalysis (version 3.0, Bruker Daltonics) considering a signal-to-noise ratio over 3, applying statistical calibration and eliminating background peaks. For identification, peaks between 850 and 1000 were not considered, as in general only matrix peaks are visible on this mass range. After processing, spectra were sent to the interface BioTools (version 3.2, Bruker Daltonics) and MASCOT search on Swiss-Prot 57.15 database was performed [Taxonomy: Homo Sapiens, Mass Tolerance 50 to 100, up to 2 miss cleavage, Global Modification: Carbamidomethyl (C), Variable Modification: Oxidation (M)]. Identification was accepted with a score higher than 56.

GLS was dissolved in 50% (v/v) MeOH to a concentration of 10 mg/mL and was applied to the MALDI stainless steel target together with 0.5 μL of 9-aminoacridine (10 mg/mL in a 1:1 mix of methanol and 0.1% (v/v) TFA, Fluka, Gillingham, Dorset, UK). The spots were allowed to dry and then analyzed in negative ion mode. Analysis was performed by MALDI-TOF/TOF mass spectrometry in the range (m/z) from 70 to 700 Da on AUTOFLEX III Smartbeam MALDI-TOF/TOF (Bruker Daltonics, Bremen, Germany). External calibration was performed using citrate (M-H (monoisotopic) 192.03 Da), glucose-6-phosphate (M-H (monoisotopic) 259.01 Da), AMP (M-H (monoisotopic) 391.19 Da), ATP (M-H (monoisotopic) 505.99 Da), and acetyl-CoA (M-H (monoisotopic) 505.99 Da) as calibrants.