Alpha cyano 4 hydroxycinnamic acid chca

Mass spectra were acquired in a positive-ion reflector mode with the use of a 4800 Plus MALDI-TOF/TOF Analyzer (Applied Biosystems, Framingham, USA). Alpha-cyano-4-hydroxycinnamic acid (CHCA) from Sigma-Aldrich (Munich, Germany), dissolved in 50:50 water/acetonitrile (J.T. Baker, Deventer, The Netherlands) with 0.1% TFA – final concentration (Sigma-Aldrich, Munich, Germany), was exploited as a MALDI matrix. External calibration was achieved with a 4700 proteomics analyzer calibration mixture provided by Applied Biosystems. Samples were spotted onto a 384 Opti-TOF MALDI plate and analyzed. Data Explorer Software, Version 4.9 was applied to process acquired spectra. Mascot Distiller Software (version 2.5.1.0, Matrix Science) was employed to predict fragment ions from given peptide sequences and overlay them on the acquired MS/MS spectra.

Urea, 3-[(3-chloromidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium fluoride (NaF), sodium orthovanadate (Na₃VO₄), ethylene diamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF) and alpha-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Sigma-Aldrich (St. Louis, MO). Trifluoroacetic acid (TFA) and acetonitrile (ACN) were obtained from Thermo Scientific (Rockford, IL).

In preparation for MALDI-TOF MS analysis, all isolated strains (Table S1) were sub-cultured on Modified Arabinose Gluconate (MAG) plates (Sadowsky et al., 1987 (link); Van Berkum, 1990 (link)) for four days to get colonies with less exopolysaccharides, facilitating the spotting of the cells on the plates for cell lysis and analysis (pers. comm. Dominik Ziegler). All these sample preparation steps were done as described in Ziegler et al., 2012 (link), Ziegler et al., 2015 (link) by Mabritec AG, Switzerland (http://www.mabritec.com), a laboratory specialised on diagnostic analyses, using MALDI-TOF MS. In brief, bacterial samples were spotted in duplicate on MALDI steel target plates. Spots were overlaid with 1 μl of 25% formic acid, air-dried, and overlaid with 1 μl of alpha-cyano-4 hydroxycinnamic acid (CHCA; Sigma Aldrich, Buchs, Switzerland) in 33% acetonitrile (Sigma Aldrich), 33% ethanol and supplemented with 3% trifluoroacetic acid (TFA). After co-crystallisation at room temperature, target plates were introduced into the MALDI-TOF Mass Spectrometer Axima™ Confidence machine (Shimadzu- Biotech Corp., Kyoto, Japan) for sample analysis.

RNF and its analogues, RNF1 and RNF3L, were synthesised using standard Fmoc amino acids on a Tribute automated peptide synthesiser (Protein Technologies Inc., Tucson, AZ, USA). When the synthesis was completed, the peptides were cleaved from the resin and de-protected. Using a weak solution of hydrogen peroxide, the samples were oxidised to form the disulphide loop structure. After purification on an Adept CECIL4200 RP-HPLC (Amersham Biosciences Inc., Piscatawa, NJ, USA), Column: Phenomenex Aeris Peptide, C18, 250 mm × 10.0 mm (Phenomenex, Macclesfeld, UK), the masses of peptide samples were confirmed by using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (Perseptive Biosystems, Framingham, MA, USA). Alpha-cyano-4-hydroxycinnamic acid (CHCA) (Sigma Chemical Co., St. Louis, MO, USA) was used as matrix and was dissolved in TFA/H₂O/acetonitrile (0.05/49.95/50, v/v/v).