C18 column

The trypsin-digested peptides were analyzed by Q Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to a nano-flow UHP-LC system (Easy-nLC1200, Thermo Fisher Scientific). Peptides (200 ng) were first loaded on a trapping column and subsequently separated on a C18 column (75 μm × 150 mm, 5 μm 200 Å, Dr. Maisch). The mobile phase consisted of a binary mixture of water and acetonitrile alone with formic acid.
The peptides were separated from 5% to 35% of solvent B in 85 min at a flow rate of 300 nl/min. The tandem mass spectra were acquired automatically by using Thermo Xcalibur software (Thermo Fisher Scientific). An information-dependent acquisition method with higher-energy C-trap dissociation (HCD) fragmentation of top 10 most intense ion in survey scan of mass range 300–1800 m/z was used. Samples were run in triplicate in randomized batches. To establish the consistent performance of the instrument, both a pooled sample and in-house standard digest were analyzed at the beginning and end of the batch.

Desalination of the peptides with TMT labels obtained from five groups was carried out using a C₁₈ column (Dr. Maisch, Germany). Next, the peptides were gathered and dried by vacuum. The 100 μg peptide samples were separated by reversed-phase HPLC (Thermo Scientific, United States) at pH = 10. Chromatographic column: 150 mm × 2.1 mm (waters, XBridge BEH C 18 XP Column); Mobile phases A: 10 mM ammonium formate aqueous solution, pH = 10; Mobile phases B: 10 mM ammonium formate, 90% ACN, 10% H₂O, pH = 10. Liquid phase gradient 120 min, mobile phase B: 5% for 2 min, 5–28% for 78 min, 28–50% for 12 min, 50–80% for 2 min, 80% for 4 min, 80–5% for 2 min and 5% for 20 min. One fraction was collected in 40 s intervals. A total of 180 fractions were collected and combined to obtain 20 fractions. The fractions were subjected to dried by vacuum and followed by storage at −80°C.

Gene variants of a clinical isolate of NCD (GenBank: CAE46584.1) were cloned into a pET-47b(+) vector, transfected into BL21 (DE3) E.coli cells (New England Biolabs). After 3 h of growth at 37 °C, recombinant expression was induced via 0.5 mM isopropyl β-D-1-thiogalactopyranoside for a total of for 3 h at 37 °C^{19 (link)}. In the sequence, the terminal (134) tryptophan residue was removed to prevent dye quenching; the only natural cysteine at position 98 was changed to methionine; and either one or two cysteine residues were introduced using site-directed mutagenesis at the desired labeling positions (S2, T65, and S130), resulting in 6 different amino acid sequences (Supplementary Table 3). The correct mass of all proteins was confirmed via electrospray ionization mass spectrometry (Functional Genomics Center Zurich). See Supplementary Table 7 for the sequences of all primers used in this work.
Immobilized metal-ion affinity chromatography and HPLC were used to purify the recombinantly expressed proteins. The proteins were then stochastically labeled with maleimide-functionalized donor and/or acceptor dyes and purified on a reversed phase C18 column (Dr. Maisch) using HPLC to remove double donor and double acceptor species. Samples were lyophilized overnight, then dissolved in 50 mM sodium phosphate buffer (PB) pH 7.0 and stored at −80 °C until use.

Measurements were performed on a Q Exactive Plus mass spectrometer (Thermo Scientific) coupled to a Thermo Scientific EASY-nLC1000 HPLC system (Thermo Scientific). Peptides were separated on self-packed C₁₈ columns (#11-1-9.aq.0001, Dr Maisch GmbH) using the following gradient: 0–157 min, 2–22% solvent B; 157–208 min, 22–40% solvent B; 208–212 min, 40–95% solvent B at a flow rate of 225 nl min⁻¹. Solvent A consisted of 0.1% formic acid, and solvent B consisted of 80% acetonitrile (#20048.320, VWR) in 0.1% formic acid (#5330020050, Millipore). Peptides were ionized with spray voltages of 2.0–2.4 kV. Data acquisition for TMT samples was performed with Xcalibur 3.1 (Thermo) in positive ion mode with a Top15 data-dependent acquisition method. The full scan was set at a resolution of 70,000 with a scan range of 300–1650 m/z and an AGC target of 3*10⁶. The MS/MS was triggered with a 1.8 m/z isolation window at a minimum intensity threshold of 8.3*10³ with the following settings: ion charge 2–7; peptide match preferred; isotope exclusion on and a dynamic exclusion set for 35 s. Fragmentation energy was set to 33, resolution at 35,000 with an AGC target of 1*10⁵ and a maximum injection time of 120 ms.