Microflex maldi tof instrument

TEFM was subjected to limitied proteolysis using trypsin (Sigma, protease/TEFM ratio 1: 20,000 w/w), ArgC protease (Promega, 1:1000 w/w), LysC protease (Sigma, 1:1000 w/w) for 1 h at RT. Products of protease digestion of TEFM were dissolved in a solution containing 5% acetonitrile and 0.1% tetrafluoroacetic acid and purified using C4 ZipTip. The peptide mixtures were analyzed using a Bruker MicroFlex MALDI-TOF instrument in sinapinic acid matrix with R=5000.

All chemicals and reagents were purchased from Sigma-Aldrich, Fisher, Aces Pharma, AmBeed, or AA Blocks and used without further purification. Flash chromatography was performed using a Teledyne CombiFlash system using prepackaged silica cartridges from Teledyne. All reactions were run under inert atmosphere unless otherwise noted. RP-HPLC was performed with a Shimadzu LC-20AB pump, Shimadzu SIL-20A HT auto sampler, and Shimadzu SPD-M20A diode array detector using Shimadzu Prominence LC. RP-HPLC was performed using a Luna^® C18 column (5μM, 250mm x 10 mm) (Phenomenex) with product eluting from a mixture of solvent A (water, 0.1 % TFA) and solvent B (acetonitrile, 0.1% TFA). TLC analysis was performed on aluminum backed 60 F₂₅₄ silica sheets from Sigma Aldrich. NMR spectra were recorded on a Bruker Ascend 700 MHz, Bruker Avance III 500 MHz, or Bruker Nanobay 400 MHz spectrometers and referenced to deuterated solvent peak. NMR spectra were processed using Mestrelab Research’s Mnova software (Santiago de Compostela, Spain). ESI-MS was performed on an Aligent 6110 single quad mass spectrometer, HRMS was performed on a Bruker Impact II QTOF mass spectrometer, and MALDI mass spectrometry was performed on a Bruker Microflex MALDI-TOF instrument. All final compounds were purified to >95% as assessed by HPLC with UV detection at λ= 213 and 353 nm.