Endoproteinase gluc

Fragments of NHLRC2 containing the Trx-like and β-propeller domains (1–575) and the C-terminal domain (587–726) for SEC-MALS analysis were prepared by cleavage of the full-length protein with endoproteinase GluC (Roche) at 1:1000 (w/w) ratio overnight at room temperature. The formation of peptide fragments of the correct length was confirmed by mass spectrometry. Purified Trx-like domain with a linker region and β-propeller domain were mixed in equimolar ratio and incubated for 3 hours at room temperature. Full-length NHLRC2, the Trx-like domain, the β-propeller domain, an equimolar mixture of the Trx-like domain and the β-propeller domain, and full-length NHLRC2 after GluC treatment were sequentially loaded onto a Superdex 200 Increase 10/300 size exclusion column (GE Healthcare) pre-equilibrated with 20 mM BisTris pH 6.5, 150 mM NaCl, 2 mM DTT. The eluted samples first passed through a miniDAWN™ TREOS multi-angle light scattering detector (Wyatt technology) and then through a RID-10A refractometer (Shimadzu). The data were analyzed using ASTRA software (Wyatt Technology).

LlTrxR (20 μg; irradiated or light-protected) in 8 M urea, 50 mM NH₄CO₃ (45 μL) was added to 2.4 μL 100 mM DTT and incubated (40 min, 21 °C), followed by addition of 2.5 μL 200 mM iodoacetamide and incubation in the dark (40 min, 21 °C). After 1:10 dilution with 50 mM NH₄CO₃, endoproteinase GluC (Roche) was added to the samples in a 1:40 enzyme:protein ratio (w/w) and incubated overnight at 29 °C. Trifluoroacetic acid was added to the digested samples to a final concentration of 0.5% and subjected to StageTip purification with empore C18 disks (3 M) as previously described39 (link). Samples (1 μg) were analysed on a Q-Exactive Orbitrap (Thermo Fisher Scientific) coupled to an EASY-nLC 1000 liquid chromatograph (Thermo Fischer Scientific). Peptides were loaded onto a custom-made nanoLC column (15 cm, C18, 100 Å, 1.9 μm particle size, 75 μm ID) packed into a Picofrit emitter (New Objectives) and eluted using a 70 min gradient at a flow rate of 250 nL min⁻¹. Resolution of 70,000, automatic gain control (AGC) value of 3·10⁶, maximum injection time (IT) of 20 ms and scan range of 300 to 1500 m/z were used for MS scans. MS/MS spectra were acquired in data-dependent mode (Top 10 method) with resolution of 17,500, AGC value of 1·10⁶, and maximum fragmentation accumulation time of 60 ms.

Cleavage of chimeric proteins into separate modules (FP and Tx) was carried out by endoproteinase Glu-C (Roche) for eGFP-OSK1 and by TEV protease (Sigma-Aldrich) for RFP-AgTx2 following the manufacturers’ guidelines. Digestion products were separated by reversed-phase HPLC.

For analysis of peptides and proteins from SILAC-labeled cells, lysates were digested using LysC and trypsin whereafter the resulting peptides mixture was desalted using Sep-Pak columns (Waters). Aliquots of 2 mg of desalted peptides were separated into 46 fractions using reversed-phase chromatography with alkaline running buffers and a Ultimate 3000 high-pressure liquid chromatography (HPLC) system (Dionex) as outlined previously^{30 (link)}. Each fraction was acidified by adding formic acid to a final concentration of 0.1% and subsequently concentrated by vacuum centrifugation. The concentration of peptides was spectrophotometrically determined at 280 nm using a NanoDrop instrument (Thermo Scientific). Peptides were then diluted in 5% acetonitrile and 0.1% trifluoroacetic acid, whereafter 1 µg of peptide was loaded for LC-MS/MS analysis.
For specific analysis of the methylation status of Lys55 and the N terminus of eEF1A samples were diluted in 2 M guanidine hydrochloride, 5 mM tris(2-carboxyethyl)phosphine, 10 mM chloroacetamide, 100 mM Tris pH 8.5 and digested with with endoproteinase Glu-C (Roche) or Chymotrypsin (Roche), respectively. The resulting peptides were desalted, stored on and eluted from C₁₈ StageTips^{65 (link)}.