Prominence lc 20ad nano hplc

For the first DIGE set, 500 μg of protein consisting of equal amounts of the cytosolic and membrane fractions of the respective T and O variant of each strain were pooled and separated according to the protocols described above (omitting the labelling reaction). After SDS-PAGE, the gels were fixed using 40% ethanol and 10% acetic acid and proteins stained afterwards using Coomassie brilliant blue. The proteins of interest were picked manually, ensuring the correct spot identity by comparison of the DIGE derived spot pattern with the spot pattern on the Coomassie brilliant blue stained gel. Liquid chromatography electrospray ionisation ion-trap mass spectrometry (LC-ESI-IT MS) using an HTC Ultra 3D ion trap (Bruker Daltonics) was performed as detailed in Supplementary Information. For the second DIGE set of experiments, the proteins of interest were excised from the DIGE gels using an Ettan Spot Picker (GE Healthcare). To account for the lower protein loading in the DIGE gels relative to that of the Coomassie stained gels, the proteins were identified using a LTQ Orbitrap mass spectrometer (Thermo Fisher). Liquid chromatography-mass spectrometry (LC-MS) with the Orbitrap was performed using a Shimadzu Prominence LC-20AD nano HPLC (Shimadzu, Japan) and Mass Spectrometer, coupled using the Nanospray Source I (Thermo Fisher Scientific) and a nanospray emitter (NewObjective, MA).

Total proteins were extracted from the TuMV-infected and control leaves. Samples were homogenized in lysis buffer solution (7 M Urea, 1 mM PMSF, 2 mM EDTA, 2 M Thiourea, 10 mM DTT, and 4% CHAPS) and were precipitated by acetone. Then, 100 μg digested proteins of each sample were labeled with different isobaric tags, and the iTRAQ tags used were as follows: CK_1-114 isobaric tag, CK_2-116 isobaric tag, CK_3-118 isobaric tag, TuMV_1-117, TuMV_2-119, and TuMV_3-121. These samples were blended and lyophilized before conducting strong cation exchange chromatography (SCX) using the Shimadzu LC-20AB HPLC system (Shimadzu, Kyoto, Japan). Each of the above components was fractionated using a Prominence LC-20AD Nano HPLC (Shimadzu, Kyoto, Japan). Finally, Q-EXACTIVE (Thermo Fisher Scientific, San Jose, CA, United States), which involves nanoelectrospray ionization followed by tandem mass spectrometry (MS/MS), was used for data acquisition. The resolution used in the Orbitrap was 70,000, and the higher-energy collision dissociation (HCD) mode (27 ± 12% collision energy) was used for MS/MS.