Ultraflex 2 maldi tof tof tandem tof mass spectrometer

The purified peptide and digested peptide fragments after each protease treatment were desalted by reversed phase chromatography using Poros Oligo R3 reversed-phase material staged in GE Loader tips. Retained peptides were eluted onto a stainless-steel target plate using 70% ACN in 1% TFA containing 5 mg/mL α-CHCA. Mass spectra were acquired on an Ultraflex II MALDI–TOF/TOF (tandem TOF) mass spectrometer controlled by flexControl software (version 2.4, Bruker Daltonics, Bremen, Germany). The instrument was operated in the positive reflector ion detection mode, and spectra were recorded in the mass range of m/z 500–4000. Typically, signals from 1000 laser shots (10 × 100 shots at 10 different positions) were averaged. Spectra were externally calibrated using a tryptic lactoglobulin. Mass spectra were analyzed with Flex-Analysis software (version 2.4, Bruker Daltonics, Bremen, Germany). For MALDI–TOF/TOF analysis, the window for precursor ion selection was set to be ±1% of the mass of the precursor ion.

The standard peptide with or without phospho-tag labeling, and the labeled standard peptide spiked in normal BSA peptides before and after TiO2 enrichment were all desalted by reversed phase Poros Oligo R3 reversed-phase material staged in GELoader tips. Retained peptides were eluted onto a stainless-steel target plate using ACN/0.1% TFA (70:30, v/v) containing 5 mg/ml α-CHCA. Mass spectra were acquired on an Ultraflex II MALDI-TOF/TOF (tandem TOF) mass spectrometer controlled by flexControl software (version 2.4, Bruker Daltonics, Bremen, Germany). The instrument was operated in the positive reflector ion detection mode, and spectra were recorded in the mass range of m/z 500–4000. Typically, signals from 1000 laser shots (10 × 100 shots at 10 different positions) were averaged. Spectra were externally calibrated using a tryptic lactoglobulin. Mass spectra were analyzed with flex- Analysis software (version 2.4, Bruker Daltonics). For MALDI-TOF/TOF analysis, the window for precursor ion selection was set to be ±1% of the mass of the precursor ion.