Easy spray

MS was performed to validate candidate molecules in the CM of Schwann cells. Briefly, RSC96 and sNF96.2 CM were denatured, alkylated, and digested in situ with trypsin. Tryptic peptides were analyzed using an Orbitrap Fusion LUMOS mass spectrometer (Thermo Fisher Scientific) coupled to an Easy-nLC 1200 via an Easy Spray (Thermo Fisher Scientific).

Analysis was by nano Liquid-Chromatography (nLC) (Ultimate 3000, Dionex) coupled to a mass spectrometer (Q Exactive, Thermo Fisher Scientific) through an EASY-Spray nano-electrospray ion source (Thermo Scientific). A pre-column (Acclaim®PepMap 100, 75 μm x 2 cm, nanoviper fitting, C18, 3 μm, 100 Å, Thermo Scientific) and analytical column (EASY-Spray Column, PepMap, 75 μm × 15 cm, nanoviper fitting, C18, 3 μm, 100 Å, Thermo Scientific) were used to trap and separate peptides, respectively. For nLC separation, buffer A was 0.1% FA and buffer B was 95% ACN/0.1% FA. A 30 minute gradient of 1% to 35% buffer B was used for peptide separation. Mass spectrometry constituted of full scans (m/z 300–1800) at a resolution of 70,000 (at m/z 200) followed by up to 10 data dependent MS/MS scans at a resolution of 17,500. HCD collision energy was 28%. Dynamic exclusion of 30 s as well as rejection of precursor ions with charge state +1 and above +8 were employed.

Samples were analyzed by nano LC-MS/MS analysis using an Orbitrap Fusion Tribrid (Thermo Fisher Scientific, San Jose, CA, USA) mass spectrometer equipped with an “EASY spray” nano ion source (Thermo Fisher Scientific, San Jose, CA, USA). The Orbitrap Fusion Tribrid (Thermo Scientific, San Jose, CA, USA) was interfaced with an UltiMate 3000 RSLC system (Dionex, Sunnyvale, CA, USA). Each sample was reconstituted with 0.1% formic acid in LC-MS-grade water (solvent A; Thermo Scientific, 85178; Rockford, IL, USA), and 5 μL was injected into a nanoviper C₁₈ trap column (3 µm, 75 µm × 2 cm, Dionex) at 3 μL min⁻¹ flow rate, and then separated with a 100 min gradient on an EASY spray C₁₈ RSLC column (2 µm, 75 µm × 25 cm), with a flow rate of 300 nL min⁻¹, and using solvent A and 0.1% formic acid in 90% acetonitrile (solvent B). The gradient was as follows: 10 min solvent A, 7%–20% solvent B for 25 min, 20% solvent B for 15 min, 20%–25% solvent B for 15 min, 25%–95% solvent B for 20 min, and eight min solvent A. The mass spectrometer was operated in positive ion mode with nanospray voltage set at 2.5 kV and source temperature at 280 °C. External calibrants included caffeine, Met-Arg-Phe-Ala (MRFA), and Ultramark 1621 (88323, Thermo Fisher Scientific^TM Pierce^TM; Rockford, IL, USA).

SDS-lysed patient and cell line samples were processed and digested according to the filter-aided sample preparation (FASP) method [23 (link),24 (link)]. All of the filter-processed samples used 20 μg of protein material. Peptides from both patient and cell line samples were cleaned up with the Oasis HLB μElution (Waters, Milford, MA, USA) protocol.4.4. Liquid Chromatography (LC)-MS Analysis.
Dried peptides were dissolved in 20 μL of 2% acetonitrile (ACN) and 0.5% formic acid (FA). Differently preserved THP-1 and Molm-13 samples were analyzed on an Orbitrap Elite mass spectrometer equipped with a nanospray Flex ion source coupled to an Ultimate 3000 Rapid Separation LC system (both from Thermo Scientific, Waltham, MA, USA). Approximately 0.5 μg peptides were pre-concentrated and separated, as previously described [5 (link)]. Patient samples without or with PBS wash(es) were analyzed on a Q Exactive HF Orbitrap mass spectrometer equipped with an Easy-Spray (Thermo Scientific) coupled to an Ultimate 3000 Rapid Separation LC system. Approximately 0.6 μg peptides were pre-concentrated on a 2 cm × 75 µm ID Acclaim PepMap 100 trapping column and separated on a 50 cm × 75 µm ID Easy-Spray PepMap RSLC analytical column (both from Thermo Scientific). Bound peptides were eluted within a 195 min run using a binary gradient with buffer A (0.1% FA in water) and buffer B (0.1% FA in ACN).

Peptides were separated by nano-liquid chromatography (EASY-nLC 1000, Thermo Fisher Scientific) coupled to a mass spectrometer (Q Exactive Plus Hybrid Quadrupole-Orbitrap, Thermo Fisher Scientific) through an EASY-Spray nanoelectrospray ion source (Thermo Fisher Scientific). The MS methods included a full MS scan at a resolution of 70,000 followed by 10 data dependent MS2 scans at a resolution of 17,500. The full MS scan range of 200–2000 m/z was selected, and precursor ions with the charge states of + 1 or greater than + 8 were excluded. Normalized collision energy of HCD fragmentation was set at 28%. Raw LC–MS/MS files were searched using PEAKS Studio version 8.5 against UniProt proteins corresponding to suborder Serpentes and the CDS database predicted by MAKER. A target-decoy approach was used to limit a false discovery rate (FDR) of the identified peptides to less than 1%. Parent and fragment monoisotopic mass errors were set at 10 ppm. Carbamidomethylation of cysteine (C) was used as a fixed modification. Oxidation (M), acetylation (protein N-term), phosphorylation (STY), and deamidation (NQ) were set as variable modifications. A maximum of 1 missed cleavage was allowed.

Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) was performed using a Waters NanoAcquity M-class system coupled to a Thermo Scientific Q Exactive HF mass spectrometer. Thermo Scientific EASY-Spray 50-cm × 75-µm ID length C18 columns were used to separate desalted peptides with a 5-30% acetonitrile gradient in 0.1% formic acid over 70 min at a flow rate of 300 nL/min. After each gradient, the column was washed with 90% buffer B (0.1% formic acid, 100% HPLC-grade acetonitrile) for 5 min and reequilibrated with 98% buffer A (0.1% formic acid, 100% HPLC-grade water) for 40 min.
MS data were acquired in data-dependent acquisition mode with an automatic switch between a full scan and 15 data-dependent MS/MS scans (TopN method). The target value for the full-scan MS spectra was 3 × 10⁶ ions in the 375 to 1,600 m/z range with a maximum injection time of 100 ms and resolution of 60,000 at 200 m/z with data collected in the profile mode. Precursors were selected using a 1.6-m/z isolation width. Precursors were fragmented by higher-energy C-trap dissociation with a normalized collision energy of 27 eV. MS/MS scans were acquired at a resolution of 15,000 at 200 m/z with an ion target value of 5 × 10⁵, maximum injection time of 50 ms, dynamic exclusion for 15 s, and data collected in the centroid mode.

Peptides from purifications were split and each analyzed by 2 replicate LC-MS/MS analyses. Samples were analyzed using an Ultimate 3000 RSLC Nano LC System (Dionex) coupled to an LTQ Orbitrap Velos hybrid mass spectrometer (Thermo Scientific) equipped with an Easy-Spray (Thermo Scientific) ion source. Peptides were desalted on-line using a capillary trap column (Acclaim Pepmap100, 100 μm, 75 μm × 2 cm, C18, 5 μm (Thermo Scientific)) and then separated using 120/180 min RP gradient (4–30% acetonitrile/0.1% formic acid) on an Acclaim Pepmap100 RSLC C18 analytical column (2 μm, 75 μm id x 50 cm, (Thermo Scientific)) with a flow rate of 0.3 μl/min. The mass spectrometer was operated in standard data dependent acquisition mode controlled by Xcalibur 2.2. The instrument was operated with a cycle of one MS (in the Orbitrap) acquired at a resolution of 60,000 at m/z 400, with the top 15 most abundant multiply-charged (2 + and higher) ions in a given chromatographic window were subjected to CID fragmentation in the linear ion trap. An FTMS target value of 1e6 and an ion trap MSn target value of 10000 were used. Dynamic exclusion was enabled with a repeat duration of 30 s with an exclusion list of 500 and exclusion duration of 60 s. Lock mass of 401.922 was enabled for all experiments.