Mass spec grade trypsin

The CuAAC reaction was set up using the Click-iT Protein Enrichment
Kit (Invitrogen). In short, 100 μL of alkyne bead slurry was
washed with 1 mL ultrapure H₂O, after which 250 μL
of concentrated medium, 250 μL of urea buffer, 500 μL
of 2× catalyst solution, and 1× CPIs were added. This was
incubated for 16–20 h at room temperature while rotating, after
which the beads were washed with 1 mL ultrapure H₂O. Next,
reduction and alkylation of the bound proteins were done by incubating
the beads with 10 mM DTT in 500 μL of SDS buffer for 15 min
while shaking, followed by incubation with 50 mM iodoacetamide (IAA)
in 500 μL of SDS buffer for 30 min while shaking in the dark.
The beads were transferred to spin columns and washed with 20 mL of
SDS buffer, 20 mL of 8 M urea in 100 mM Tris, pH 8, 20 mL 20% isopropanol,
20 mL 20% acetonitrile, and 5 mL of PBS. The bound proteins were digested
by resuspending the beads in 200 μL of freshly prepared digestion
buffer (2 M Urea, 100 mM Tris-HCl pH 8, 100 mM DTT) with 0.5 μg
of mass spec-grade trypsin (Promega) and overnight incubation at room
temperature while shaking. The digest was desalted and concentrated
on C18 StageTips without acidification.^{24 (link)} Peptide labeling was done by dimethyl labeling,^{27 (link)} and StageTips were stored at 4 °C until measurement
by LC-MS/MS.

Whole-cell protein extracts
were prepared by incubating organoid pellets with SDS lysis buffer
(4% SDS, 1 mM DTT, 100 mM Tris-HCl pH 7.5 in ultrapure H₂O) for 5 min at 95°C. Samples were sonicated until homogeneous
using alternating cycles of 30 s on/30 s off on high intensity and
spun down at 16 000 for 5 min. The supernatant was transferred
to a new tube and protein concentrations were determined using a Pierce
BCA protein assay (Thermo Fisher). Afterward, the final DTT concentration
was corrected to 100 mM. The organoid lysates were digested with mass
spec-grade trypsin (Promega) using filter-aided sample preparation
(FASP^{22 (link)}) and subsequently fractionated
using strong anion exchange (SAX^{23 (link)}). The
flow through, pH 11, pH 8, pH 5, and pH 2 fractions were collected.
The fractions were desalted and stored on StageTips at 4 °C until
measurement by LC-MS/MS.^{24 (link)}

All materials were from Sigma unless otherwise noted. ActivX desthiobiotin ATP kinase enrichment kit and BCA assay were from Pierce. Mass spec grade trypsin was from Promega. Amicon Ultra Centrifugal Filters and C18 ziptips were from Millipore. Bondbreaker TCEP, 5 mL 7 K MWCO Zeba spin desalting columns, and formic acid were from ThermoFisher. Oasis HLB 1 cc extraction columns were from Waters. Lysis buffer for phosphorylation analysis was from Cell Signaling Technologies. Sequencing grade trypsin was from Promega. Ni-NTA agarose beads were from Qiagen. MEM (11095-072) and Penicillin-Streptomycin (15140122) for cell culture were from ThermoFisher Scientific. Fetal Bovin Serum for cell culture was obtained from R&D Systems (S111560). The antibodies used in the immunoassays are as follows: ASS1(Polaris), ERK1/2 (CST 4695), phospho-ERK1/2 (Thr202/Tyr204) (CST 4370), cMyc (Abcam ab11917), phospho-cMyc (S62) (abcam ab51156).

Tomato fruits (Piccadilly variant) were purchased in the local market (G.M Fruit, Sicily, Italy). Sodium hydrogen phosphate dihydrate (Na₂HPO₄·2H₂O), sodium dihydrogen phosphate (NaH₂PO₄·H₂O) and sodium hydroxide (NaOH) were from J.T. Baker (Deventer, The Netherlands). Leupeptine, phenylmethylsulfonyl fluoride (PMSF), sodium azide, sucrose and colloidal Coomassie Brilliant blue G-250 were obtained from AppliChem (Darmstadt, Germany). OptiPrep™ (60% (w/v)) was from Serumwork (Bernburg AG, Germany). Sepharose Cl-2B, sucrose and Triton^® X-100 were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). RapiGest detergent was obtained from Waters Corporation (Milford, MA, USA). Trypsin (Mass Spec grade) was from Promega Corporation (Madison, WI, USA). Qubit Protein Assay Kit was from Thermo Fisher Scientific (Rockford, IL USA). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) buffers, reagents and materials: Novex Bolt 4–12% Bis-Tris Plus gel and Bolt MOPS SDS running buffer were from Invitrogen (Carlsbad, CA, USA). Water (18.2 MΩ.cm (25 °C), 0.22 µm filtered) generated by a MilliQ system (Merck Millipore) was used. Other solvents used for proteomics were LC–MS grade (VWR International, Debrecen, Hungary). Leucine enkephalin peptide (amino acid sequence is YGGFL) was purchased from Waters Corp. (Wilmslow, UK).

Tubulin bands were excised from the coomassie stained gel, after which proteins were reduced with dithiothreitol and alkylated with iodoacetamide. Proteins were digested with trypsin (mass spec grade, Promega) overnight at 37°C and peptides were extracted with acetonitrile. Digests were dried in a vacuum centrifuge and reconstituted in 10% formic acid for MS analysis. Peptide mixtures (10% of total digest) were loaded directly on the analytical column and analyzed by nanoLC-MS/MS on an Orbitrap Fusion Tribrid mass spectrometer equipped with a Proxeon nLC1000 system (Thermo Scientific) as described previously [39 (link)]. Solvent A was 0.1% formic acid/water and solvent B was 0.1% formic acid/80% acetonitrile. Peptides were eluted from the analytical column at a constant flow of 250 nl/min in a 90-min gradient, containing a 74-min linear increase from 5% to 24% solvent B, followed by a 16-min wash at 80% solvent B.