Proteomics grade trypsin

The FgPRP4-3xFLAG fusion construct was generated by the gap repair approach by co-transformation of the full-length FgPRP4 fragment and XhoI-digested pFL7 into yeast strain XK1-25 [48 (link)]. The resulting fusion construct rescued from Trp⁺yeast transformants was confirmed by sequence analysis and transformed into the wide-type strain PH-1. Geneticin-resistant transformants expressing the fusion constructs were identified by PCR and confirmed by western blot analysis with the anti-FLAG antibody (Sigma). Total proteins isolated from the resulting transformant were incubated with the anti-FLAG M2 beads (Sigma) as described [54 (link)]. Proteins eluted from anti-FLAG beads were digested with proteomics grade trypsin (Sigma) and enriched for phosphopeptides with the polymer-based metal ion affinity capture (PolyMAC) as described [29 (link)]. Phosphopeptides enriched by PolyMac were analyzed with an ABI 4800 MALDI-TOF/TOF mass spectrometer. Proteome Discoverer (version 1.0; Thermo Fisher Scientific) was used to identify peptide sequences and phosphorylation sites as described [29 (link)].

Each sample was diluted with loading sample buffer and then applied onto 1.2-cm wide wells of a conventional SDS-PAGE gel (1mm-thick, 4% stacking, and 12% resolving) (Figure S1). Then run was stopped as soon as the front entered 1 cm into the resolving gel, so that the whole proteome became concentrated in the stacking/resolving gel interface. The unseparated protein bands were visualized by Coomassie staining, excised, cut into cubes (1 mm²), deposited in 96-well plates and processed automatically in a Proteineer DP (Bruker Daltonics, Bremen, Germany). The digestion protocol used was based on [81 (link)] with minor variations: gel plugs were washed firstly with 50 mM ammonium bicarbonate and secondly with ACN prior to reduction with 10 mM DTT in 25 mM ammonium bicarbonate solution, and alkylation was carried out with 55 mM IAA in 50 mM ammonium bicarbonate solution. Gel pieces were then rinsed firstly with 50 mM ammonium bicarbonate and secondly with ACN, and then were dried under a stream of nitrogen. Proteomics Grade Trypsin (Sigma Aldrich) at a final concentration of 16 ng/μL in 25% ACN/50 mM ammonium bicarbonate solution was added and the digestion took place at 37 °C for 4h. The reaction was stopped by adding 50%ACN/0.5%TFA for peptide extraction. The tryptic eluted peptides were dried by speed-vacuum centrifugation.

Beads were processed for mass spectrometry as follows: they were eluted with Laemmli buffer and samples loaded into an SDS-PAGE gel (1 mm thick; 4% stacking; 12% resolving), which was run briefly, until the front entered 1 cm into the resolving gel (so the whole proteome was concentrated at the stacking-resolving gel interface). The unresolved protein bands were then visualized with Coomassie, excised, cut into cubes (1 mm³), deposited into 96-well plates, and processed automatically in a Proteineer DP (Bruker Daltonics). Digestion protocol was as described (Shevchenko et al., 1996 (link)), with minor variations: gel plugs were first washed with 50 mM NH₄HCO₃, then with acetonitrile prior to reduction with 10 mM dithiotreitol (DTT) in 25 mM NH₄HCO₃ solution. Alkylation was performed in 55 mM iodoacetamide in 50 mM NH₄HCO₃. Gel pieces were then rinsed in 50 mM NH₄HCO₃, then in acetonitrile, then dried under a nitrogen stream. Digestion was carried out for 4 h at 37°C using 16 ng/μL proteomics-grade Trypsin (Sigma), in 25% acetonitrile in 50 mM NH₄HCO₃. Reaction was stopped with 0.5% trifluoroacetic acid in 50% acetonitrile. The eluted tryptic peptides were dried by speed-vacuum centrifugation.