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5 protocols using dl dtt

1

Quantitative Proteomics Sample Preparation

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A total of 27 TIF fluid samples were supplemented with urea to reach a final concentration of 6 mol/l and then quantified with the colorimetric RC DCTM protein assay quantification kit. Twelve micrograms of protein of each sample were digested with Lys-C and Trypsin. Prior to digestion, samples were reduced with 10 mM DL-DTT (Sigma-Aldrich, P/N D9163-25G) and alkylated with 55 mM IAA (Sigma-Aldrich, P/N C0267); then the samples were diluted with Tris 0.1 M to reach urea 2 mol/l. Lys-C (Wako Chemicals) was added at 1:25 (w/w) (enzyme-to-protein ratio), and protein digestion was carried out at 30 °C for 16 h. The samples were subsequently diluted again with Tris 0.1 M to reach urea 0.8 mol/l. Trypsin (Promega, P/N V5117) was added at 1:25 (w/w) (enzyme-to-protein ratio), and protein digestion was carried out at 30 °C for 8 h. The enzymatic reaction was stopped with formic acid (FA; Sigma-Aldrich, P/M 1.00264.0100) (10% (v/v) final concentration). The digested samples were desalted using C18 microspin column (The Nestgroup). Desalted peptides were dried in the speedvac.
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2

Proteomic Analysis of CPMV Capsid Subunits

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The capsid protein subunits (L and S) of CPMV virus particles from Cowpea and CPMV eVLPs were separated on an SDS-PAGE gel as previously described (Sainsbury et al., 2011 (link)). The S subunit of CPMV virions and that of eVLPs contained two bands corresponding to the slow and fast forms (Figures 5A and 5B). The bands corresponding to the slow and fast forms of the S subunit of eVLPs and of the fast form (only) of the virions were excised and submitted for proteomics mass spectrometry analysis by digestion with GluC protease. The excised gel bands were destained with a mixture of a 50:50 (v/v) dilution of acetonitrile-25 mM ammonium bicarbonate and dried with a SpeedVac system. The protein samples in the gel were then reduced in 25 μl of 10 mM d,l-DTT (Sigma) for 1 hr and alkylated with 25 μl of 55 mM iodoacetamide (Sigma) for 30 min in the dark prior to an 18-hr GluC digestion at 37°C using a 1:30 (w/w) enzyme-to-substrate ratio. The resulting peptides were extracted twice with a 50:45:5 (v/v) volume of acetonitrile-water-formic acid and concentrated to 30 μl before being analyzed. Peptides were analyzed by reverse-phase chromatography prior to mass spectrometry analysis and tandem mass spectrometry samples were analyzed by using Mascot (version 2.1.04; Matrix Science) as described in Powers et al. (2011) (link).
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3

Protein Separation and Digestion Protocol

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Acetonitrile, methanol, formic acid (FA), trichloroacetic acid, water (HPLC grade), 16% Tricine gel, and Tricine SDS running buffer were from Thermo Fisher Scientific. Acetic acid (AA), ethanol, and chloroform were from DUKSAN. Lysyl endopeptidase (Lys-C, mass spectrometry grade) and trypsin (sequencing grade) were purchased from Promega. Ammonium formate, ammonium bicarbonate, DL-DTT, and iodoacetamide were from Sigma-Aldrich, and all other reagents were from Sigma-Aldrich.
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4

WRN Protein Stabilization Assay

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Cells were cultured in a 225 cm2 flask (Falcon, 353138) to a maximum confluency of 95%, then cells were rinsed twice in PBS and lysed in 12.5 ml of lysis buffer per flask (50 mM Tris pH 7.8, 1% NP40 (Sigma-Aldrich, 74385), 120 mM NaCl (Sigma-Aldrich, 71380), 25 mM NaF (Merck, 1.06450.0025), 40 mM β-glycerophosphate disodium salt (Sigma-Aldrich, 50020), 100 µM sodium metavanadate (Sigma-Aldrich, 590088), 1 mM DL-DTT (Sigma-Aldrich, 43815), 100 µM phenylmethyl sulfonyl fluoride (Sigma-Aldrich, P-7626), 1 mM benzamidine (Sigma-Aldrich, B-6506), 1 µM microcystin (Alexis Biochemicals 350-012-M001)). The lysates were centrifuged at 4 °C for 10 min and total protein was quantified and adjusted to 0.5 µg µl−1. Then, 100 µl of cell lysates was then plated in 96-well plates, the lysates were treated with HRO761 starting at 10 µM using the HP Dispenser (software D300eControl) and then incubated for 72 h at 20 °C. After incubation, the plates were used for in WRN enzyme-linked immunosorbent assay (ELISA) analysis as described below. PS50 values are the levels of HRO761 needed to stabilize the WRN protein 50% over DMSO control.
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5

Mass Spectrometry Glycoproteomic Protocol

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Sodium borohydride, sodium chloride, Dowex cation-exchange resin (50W-X8), ammonium bicarbonate (ABC), TFA, Dulbecco’s PBS (DPBS), hydrochloric acid (HCl), and dl-DTT were purchased from Sigma–Aldrich. Ethanol (Reag. Ph. Eur) and bovine submaxillary mucin (BSM), type I-S, were purchased from Merck. Tandem mass tag (TMT)pro label reagents, 8 M guanidine hydrochloride, Dulbecco’s modified Eagle’s medium, 0.25% trypsin/EDTA, and fetal calf serum (FCS) were obtained from Thermo Fisher Scientific. Potassium hydroxide was obtained from Honeywell Fluka. Solid phase extraction bulk sorbent carbograph was obtained from Grace Discovery Sciences. HPLC SupraGradient acetonitrile (methyl cyanide [MeCN]) was obtained from Biosolve. Peptide N-glycosidase F (PNGase F) and complete EDTA-free protease inhibitor cocktail tablets were purchased from Roche Diagnostics. A 96-well PP filter plate was purchased from Orochem Technologies. MultiScreen HTS 96 multiwell plates (hydrophobic Immobilon-P polyvinylidene difluoride [PVDF] membrane) and 96-well PP microplate were obtained from Millipore.
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