4800 maldi tof tof ms

Samples were analyzed using a 4800 MALDI-TOF/TOF MS (AB/Sciex, Framingham, MA). For quantitation, spectra were acquired in positive reflector mode with a fixed laser intensity between 2800 and 3500 arbitrary units. To confirm the identity of the target peaks, MS/MS spectra were acquired in positive mode using post-source decay for fragmentation. Spectra were acquired using 4000 Explorer software and exported to Data Explorer (AB/Sciex, Framingham, MA). Mass calibration was performed using internal trypsin autolysis peaks or external calibrants (Sigma, St. Louis, MO). Quantitation on peaks with a signal-to-noise ratio greater than 3 or isotope clusters with a combined signal-to-noise ratio greater than 10 in the relevant mass ranges was performed in Excel software (Microsoft). Target peptides were quantified by comparing their peak area to the corresponding labeled peptide [32 (link)].

The reagents and solvents were purchased from commercial sources (Fisher) and used directly. The intermediates and products were purified by using an ISCO CombiFlash system and prepacked SiO₂ cartridges. Final compounds were purified on preparative high-pressure liquid chromatography (RP-HPLC) was performed on Agilent 1260 Series system. Systems were run with 0–20% methanol/water gradient with 0.1% TFA. NMR spectra were acquired on a Bruker AV500 instrument (500 MHz for ¹H-NMR, 126 MHz for ¹³C-NMR). TLC-MS were acquired using Advion CMS-L MS. Matrix-assisted laser desorption ionization mass spectra (MALDI-MS) data were acquired in positive-ion mode using a Sciex 4800 MALDI TOF/TOF MS. The peptides were synthesized using a CEM Liberty Blue Automated Microwave Peptide Synthesizer with the manufacturers standard coupling cycles at 0.1 mmol scale. The purity of final compounds was confirmed by Waters LC-MS system and/or Agilent 1260 Series HPLC system. Systems were run with 0–40% methanol/water gradient with 0.1% TFA. All the purity of target compounds showed >95% in RP-HPLC.