C18 sep pack column

Peptide was labelled as previously described^{38 (link)}. Briefly, ⁶⁴Cu was prepared following the ⁶⁴Ni(p,n)⁶⁴Cu reaction using an enriched ⁶⁴Ni target electroplated on a rhodium disk and converted to [⁶⁴Cu]Cu[II] acetate ([⁶⁴Cu]Cu(OAc)₂) by dissolving the [⁶⁴Cu]CuCl₂ in ammonium acetate (0.1 M; pH 5.5). The peptide (5 μg) was dissolved in ammonium acetate buffer (0.1 M, pH 5.5) with [⁶⁴Cu]Cu-(OAc)₂ (300–370 MBq; 8–10 mCi) in a total volume of 300–350 μL. The resulting solution was incubated at 95 °C for 10 min and unchelated ⁶⁴Cu was removed on a C18 Sep-Pack Column (Waters, USA). The peptide was collected, evaporated and counted in a Capintec radioisotope calibrator (Capintec, Inc., USA) to calculate the specific activity of the product. The resulting ⁶⁴Cu-peptide was reconstituted in PBS at pH 7.4 and used for conjugation with MSA.

Total IAA, ABA, GA3, and ZR content in NE and E samples at each time point were extracted and purified using the following method. Fresh tissues (0.5–1.0 g) were ground to a powder with a mortar and pestle in liquid nitrogen. The samples were extracted in 2 mL methanol containing 0.01% (w/v) butylated hydroxytoluene overnight at 4 °C. Then, the sample was centrifuged (1370× g, 8 min), and the supernatant was collected and dried under a stream of air. Samples were purified using a C-18 Sep Pack column (Waters, Milford, MA, USA) that was flushed with 1 mL 100% (v/v) methanol followed by 1 mL 10% (v/v) methanol. The samples were dissolved in 10% methanol and loaded onto the column. The column was washed with 1 mL 10% (v/v) methanol twice and flushed. Samples were eluted with 1 mL 80% (v/v) methanol and dried under a stream of air. The samples were dissolved in methanol (sample from 1 g fresh tissue was dissolved in 2 mL methanol) and diluted with Tris-buffered saline. Indirect enzyme-linked immunosorbent assays (ELISA) were used to assay phytohormones according to Norman et al. [12 (link)] for ABA, and Yang et al. [13 (link)] for IAA, GA, and ZR. The concentrations of each phytohormone were calculated based on a standard curve and expressed as ng g⁻¹ fresh weight.

Sample fractionation using strong cation-exchange liquid chromatography (SCX-LC) with a PolySULFOETHYL A column (PolyLC, 200 × 4.6 mm) was conducted with a gradient of 10–60% buffer SCX-B (1 M KCl, 20 mM Na₂HPO₄, 20% ACN, pH 2.9). Five fractions were collected as shown in Fig. 2C. For the MS analysis of the raw extract, an aliquot of the non-treated raw extract was fractionated by SCX-LC under the same conditions. Fractionated peptide mixtures were desalted using a C-18 Sep-pack column (50 mg, Waters) and lyophilized.

The remaining digested protein sample was desalted on C18 Sep-Pack column (Waters, Bedford, MA, USA) using 80% acetonitrile, 20% water with 0.1% formic acid for elution. The eluted peptides were dried in the Speed-Vac and labeled with TMT 10-plex labelling (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions.
To normalize all the samples, a pool was created by mixing an equally small aliquot of each sample and then, 40 μg of this pool and 40 μg of each of the nine individual samples were labeled. Next, a small aliquot of 5 μg from each labeled sample was mixed and purified using C18 zip-tip (Millipore, Burlington, MA, USA) and analyzed by nanoLC-Orbitrap to check the labeling reaction.