Leibniz Institute - ATI in Ancient Wheats

Leibniz Institute - ATI in Ancient Wheats
ATI in Ancient Wheats

  • Organism: Triticum
  • Instrument: TSQ Vantage,LTQ Orbitrap Velos
  • SpikeIn: Yes
  • Keywords: Amylase/trypsin-inhibitor, wheat, spelt, emmer, einkorn
  • Lab head: Katharina Scherf
Abstract
α-Amylase/trypsin-inhibitors (ATIs) were identified as triggers of intestinal inflammation and of symptoms typical of non-celiac gluten sensitivity. Studies on ATI bioactivity using monocytes suggested that the ancient wheat species spelt, emmer and einkorn may exhibit lower inflammatory activity compared to modern common wheat and durum wheat, but the absolute contents of ATIs were not assessed. Therefore, the aim of this study was to develop a method to quantitate the concentrations of the predominant ATIs 0.19+0.53, 0.28, CM2, CM3 and CM16 in eight cultivars each of five wheat species grown under the same environmental conditions. ATI-specific marker peptides were quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and stable isotope dilution assays (SIDA) using 13C- and 15N-labelled peptides as internal standards. The SIDA showed good precision, recovery, limits of detection and we confirmed the validity of our quantitative results by comparison to a label-free and untargeted LC-MS/MS analysis, in which we estimated absolute protein concentrations using the intensity based absolute quantitation algorithm (iBAQ). Spelt and emmer had higher overall ATI contents than common wheat, with durum wheat in between, whereas only three of eight einkorn cultivars contained ATIs in very low concentrations. The distribution of ATI types was suitable to distinguish hexaploid, tetraploid and diploid wheat species and could serve as species-specific fingerprint. The results point to a better tolerability of einkorn for non-celiac gluten sensitivity (NCGS) patients, because of very low overall ATI contents, but these findings need to be verified by measurements of in vitro and in vivo bioactivity.
Experiment Description
ATI Reference: An Eksigent nanoLC-Ultra 1D+ system (Eksigent Technologies, Dublin, CA, USA) was coupled online to a LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific). Using a flow rate of 5 µl/min of solvent A1 (0.1 % FA in water), peptides were loaded onto a trap column (ReproSil-Pur 120 ODS-3, 5 µm, 2 cm × 75 µm, Dr. Maisch, Ammerbuch-Entringen, Germany). Subsequently, peptides were separated on an analytical column (ReproSil-Gold 120 C18, 3 µm, 40 cm × 75 µm, Dr. Maisch) using a flow rate of 300 nl/min and a gradient from 2 % to 32 % solvent B (solvent A: 0.1 % FA and 5 % DMSO in water, solvent B: 0.1 % FA and 5 % DMSO in acetonitrile) in 110 min. The eluate from the analytical column was sprayed via a stainless steel emitter (ThermoFisher Scientific) into the MS at a source voltage of 2.2 kV. The transfer capillary was heated to 275 °C. The Orbitrap Velos was set to data-dependent acquisition in positive ion mode, automatically selecting the 20 most intense precursor ions from the preceding full MS (MS1) spectrum with an isolation width of 2.0 m/z for fragmentation using CID at 35% normalized collision energy and subsequent identification by MS/MS (MS2). MS1 (360 - 1300 m/z) spectra were acquired in the Orbitrap using a resolution of 60,000 (at 400 m/z) and MS2 spectra in the ion trap. iBAQ Data for Grain Samples: An Eksigent nanoLC-Ultra 1D+ system (Eksigent Technologies, Dublin, CA, USA) was coupled online to a LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific). Using a flow rate of 5 µl/min of solvent A1 (0.1 % FA in water), peptides were loaded onto a trap column (ReproSil-Pur 120 ODS-3, 5 µm, 2 cm × 75 µm, Dr. Maisch, Ammerbuch-Entringen, Germany). Subsequently, peptides were separated on an analytical column (ReproSil-Gold 120 C18, 3 µm, 40 cm × 75 µm, Dr. Maisch) using a flow rate of 300 nl/min and a gradient from 2 % to 32 % solvent B (solvent A: 0.1 % FA and 5 % DMSO in water, solvent B: 0.1 % FA and 5 % DMSO in acetonitrile) in 110 min. The eluate from the analytical column was sprayed via a stainless steel emitter (ThermoFisher Scientific) into the MS at a source voltage of 2.2 kV. The transfer capillary was heated to 275 °C. The Orbitrap Velos was set to data-dependent acquisition in positive ion mode, automatically selecting the 20 most intense precursor ions from the preceding full MS (MS1) spectrum with an isolation width of 2.0 m/z for fragmentation using CID at 35% normalized collision energy and subsequent identification by MS/MS (MS2). MS1 (360 - 1300 m/z) spectra were acquired in the Orbitrap using a resolution of 60,000 (at 400 m/z) and MS2 spectra in the ion trap. Dynamic exclusion was set to 60 s. SRM Data of Ancient Wheats: An UltiMate 3000 HPLC system (Dionex, Idstein, Germany) coupled to a triple-stage quadrupole mass spectrometer (TSQ Vantage, ThermoFisher Scientific, Bremen, Germany) was used. An Aqua®-C18 column (50 × 2 mm, 5 µm, 12.5 nm, Phenomenex, Aschaffenburg, Germany) was used for peptide separation with the following LC conditions: solvent A, FA (0.1%, v/v) in water, solvent B, FA (0.1%, v/v) in acetonitrile; gradient, 0 - 5 min 10% B, 5 - 20 min 10 - 90% B, 20 - 23 min 90% B, 23 - 25 min 90 - 10% B, 25 - 40 min 10% B; flow rate, 0.2 mL/min; injection volume, 10 μL; column temperature, 22 °C. The ion source was operated in the ESI positive mode and the following source parameters were set: spray voltage, 4500 V; vaporizer temperature, 50 °C; sheath gas pressure, 40 arbitrary units (au); aux gas pressure, 5 au; capillary temperature, 300 °C.
Sample Description
ATI Reference: The ATI reference was from Sigma-Aldrich (α-Amylase Inhibitor from Triticum aestivum (wheat seed), type I). iBAQ Data for Grain Samples: The cultivars each of common wheat, spelt, durum wheat, emmer and einkorn were cultivated by the State Plant Breeding Institute, University of Hohenheim (Stuttgart, Germany) at Seligenstadt, Germany, and harvested in 2013. SRM Data of Ancient Wheats: Eight cultivars each of common wheat, spelt, durum wheat, emmer and einkorn were cultivated by the State Plant Breeding Institute, University of Hohenheim (Stuttgart, Germany) at Seligenstadt, Germany, and harvested in 2013. The grains were milled into wholemeal flours using a cross-beater mill (Perten Instruments, Hamburg, Germany).
Created on 10/10/18 10:16 AM
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates
2017_Samples_Einkorn_2018-08-01_08-01-22.sky.zip (2 MB)2018-10-1057144229
2017_Samples_Emmer_2018-08-01_08-00-35.sky.zip (3 MB)2018-10-1057144231
2017_Samples_Spelt_2018-08-01_07-54-34.sky.zip (2 MB)2018-10-1057144229
2017_Samples_CommonWheat_2018-08-01_07-53-46.sky.zip (3 MB)2018-10-1057144231
2017_Samples_DurumWheat_2018-08-01_07-55-26.sky.zip (2 MB)2018-10-1057144227
ATI_10Samples_2018-08-01_08-19-31.sky.zip (8 MB)2018-10-10625267830
ATI_Standard_01_2018-08-01_09-57-03.sky.zip (7 MB)2018-10-10630341023