Finnigan lcq deca ion trap

Monolayer (2D) and tumor spheroid (3D) lysates from patient-derived primary cells were separated and analyzed using nanoACQUITY UPLC system (Waters, Milford, MA, USA) directly coupled to a Finnigan LCQ DECA ion trap mass spectrometer (Thermo Fisher). The samples were analyzed using a MS/MS spectra system (Thermo Quest, San Jose, CA, USA) and processed using SEQUEST software purchased from Thermo Fisher.

After desalting, the eluted tryptic peptides were separated and analysed using a nano ACQUITY UPLC (Waters, Milford, MA, USA) directly coupled to a Finnigan LCQ DECA iontrap mass spectrometer (Thermo Scientific, Waltham, MA, USA). In brief, the peptides were bound to the ACQUITY UPLC peptide BEH C18 column (1.7 μm size, 130Å pore size, 100 μm × 100 mm) with distilled water containing 0.1% (v/v) formic acid and the bound peptides were eluted with a 40 min gradient of 0–90% (v/v) ACN gradient with 0.1% (v/v) formic acid at a flow rate of 0.4 μL/min. For tandem mass spectrometry, the full mass scan range mode was m/z = 400–2000 Da. After determination of the charge states of an ion on zoom scans, the product ion spectra were acquired in the MS/MS mode with relative collision energy of 55%. The individual spectra from MS/MS were processed using SEQUEST software (Thermo Quest, San Jose, CA, USA) and the generated peak lists were used to query the NCBI database using the MASCOT program (Matrix Science., London, UK). We set the modifications of methionine and cysteine for MS analyses. The tolerance of the peptide mass was 2 Da. The MS/MS ion mass tolerance was 1 Da, allowance of missed cleavage was 1, and the charge states (+1, +2, and +3) were taken into account for data analyses. We considered only significant hits as defined by MASCOT probability analyses.