Autoflex 2 maldi tof tof mass spectrometer

The reaction mixtures of peptides or proteins were analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry with an Autoflex II MALDI-TOF/TOF mass spectrometer equipped with a nitrogen pulsed laser (Bruker). In brief the peptide mixtures were loaded on C18 Zip Tips separately, washed with 0.1%TFA, eluted with 2, 5-dihydroxybenzoic acid solution (Agilent), then spotted on a Bruker MTP 384 massive stainless steel MALDI target. The matrix spots were allowed to dry at room temperature. Peptide Mass Spectra were acquired in positive reflectron mode with pulsed ion extraction.

Peptide samples were spotted on a steel target plate (Bruker Daltonics, Bremen, Germany) and allowed to dry at room temperature. Matrix solution (3 mg α-cyano-4-hydroxycinnamic acid in 1 ml of 50% ACN containing 0.1% trifluoroacetic acid) was then added. MS was performed on an Autoflex II MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) using a solid nitrogen laser (337 nm) and FlexControl software in reflectron mode with positive ion mass spectra detection. The mass spectrometer was externally calibrated with Peptide Calibration Standard II (Bruker Daltonics). Spectra were acquired in the mass range 800–3,000 Da. The peak lists were generated using FlexAnalysis and searched against Swiss-Prot (2012_07 version, 536 789 sequences) using Mascot software. The peptide mass tolerance was set to 100 ppm, taxonomy Homo sapiens, missed cleavage was set to 1, fixed modification for cysteine carbamidomethylation, and variable modifications for methionine oxidation and protein N-terminal acetylation. Proteins with Mascot score over the threshold 56 for p < 0.05 calculated for the used settings were considered as identified. If the score was lower, the identity of protein candidate was confirmed by MS/MS.