C18 pepmap100

Mass spectrometry (MS) was performed using a Q-Star XL nanospray quadrupole/time-of-flight tandem mass spectrometer, nanospray-Qq-TOF-MS/MS (Applied Biosystems, Villebon-sur-Yvette, France) coupled to an online nanoLC system (Ultimate Famos Switchos from Dionex, Amsterdam, The Netherlands). One microliter of each sample was loaded onto a trap column (PepMap100 C18; 5 μm; 100 Å; 300 μM x 5 mm; Dionex), washed for 3 minutes at 25 μL/min with 0.05% trifluoroacetic acid/2% acetonitrile, then eluted onto a C18 reverse phase column (PepMap100 C18; 3 μm; 100 Å; 75 μM x 150 mm; Dionex). Peptides were separated at a flow rate of 0.300 μL/min with a linear gradient of 5–80% acetonitrile in 0.1% formic acid over 120 minutes. MS data were acquired automatically using ANALYST QS 1.1 software (Applied Biosystems). Following an MS survey scan over m/z 400–1600 range, MS/MS spectra were sequentially and dynamically acquired for the three most intense ions over m/z 65–2000 range. The collision energy was set by the software according to the charge and mass of the precursor ion. MS and MS/MS data were recalibrated using internal reference ions from a trypsin autolysis peptide at m/z 842.51 [M + H]⁺ and m/z 421.76 [M + 2H]²⁺.

Twenty visceral ganglia were extracted in 0.1% trifluoroacetic acid (TFA) at 4 °C and centrifuged for 30 min at 35,000× g at 4 °C. The supernatants were concentrated on ChromafixC18 solid phase extraction cartridges (Macherey-Nagel, Hoerdt, France). Then, the recovered samples were evaporated. For nano-LC fragmentation, peptide samples were first desalted and concentrated onto a µC18 Omix (Agilent, Technologies Co., Ltd., Palo Alto, CA, USA) before analysis. The chromatography step was performed on a NanoElute (Bruker Daltonics, Billerica, MA, USA) ultra-high pressure nano flow chromatography system. Approximatively 200 ng of each peptide sample was concentrated onto a C18 pepmap 100 (5 mm × 300 µm i.d.) precolumn (Thermo Scientific, Waltham, MA, USA) and separated at 50 °C onto a reversed phase Reprosil column (25 cm × 75 μm i.d.) packed with 1.6 μm C18-coated porous silica beads (Ionopticks, St, Fitzroy, VIC, Australia). Mobile phases consisted of 0.1% formic acid, 99.9% water (v/v) (A), and 0.1% formic acid in 99.9% ACN (v/v) (B). The nanoflow rate was set at 400 nL/min, and the gradient profile was as follows: from 2 to 15% B within 60 min, followed by an increase to 25% B within 30 min, and further to 37% within 10 min, followed by a washing step at 95% B and re-equilibration.

An amount of 5 µg peptide lysate was injected into nanoHPLC (UltiMate 3000 RSLCnano, Dionex, Thermo Fisher Scientific, Waltham, MA, USA). Peptide separation was performed on a C18-reverse-phase trapping column (C18 PepMap100, 300 µm × 5 mm, particle size 5 µm, nano viper, Thermo Fischer Scientific, Waltham, MA, USA), which was followed by a C18-reverse-phase analytical column (Acclaim PepMap^® 100, 75 µm × 25 cm, particle size 3 µm, nanoViper, Thermo Fischer Scientific). Mass spectrometric analysis of peptides was performed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a TriVersa NanoMate (Advion, Ltd., Harlow, UK) source in the liquid chromatography (LC) chip coupling mode. LC gradient, ionization mode, and the mass spectrometry mode were described [37 (link)].

For the identification of phosphorylation sites, nanoLC-MS/MS was performed on an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) coupled to a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with nano ESI source. The nanoLC system was equipped with a trap column (C18 PepMap 100, 0.3 mm × 5 mm, 5 μm, Thermo Fisher Scientific) and an analytical column (NTCC-360/75-3-125, Nikkyo Technos, Tokyo, Japan). Peptides separation was performed using 60 min gradient of water containing 0.1% FA (mobile phase A) and acetonitrile containing 0.1% FA (mobile phase B) at a flow rate of 300 nL/min. The elution gradient was set as follows: 0–3 min, 2–2% B; 3–63 min, 2–40% B; 63–65 min, 40–95% B; 65–75 min, 95–95% B; 75–77 min, 95–2% B; 77–90 min, 2–2% B. Mass spectrometer was operated in data-dependent acquisition mode.

Nano LC–MS/MS analysis was carried out using an Ultimate 3000 nanoRSLC system (Thermo Scientific) coupled in-line with an Orbitrap Fusion Tribrid™ mass spectrometer (Thermo Scientific). Then, 1 µg of digested protein samples were loaded onto a C18 trap column (C18 PepMap100, 300 μm × 5 mm, 5 μm particle size, 100 Ǻ pore size; Thermo Scientific) and desalted for 3 min using a flow rate of 25 μL/min in 0.1% (v/v) TFA, 2% (v/v) ACN as previously described by Di Luca et al. [10 (link)].

Protein digests were analyzed by a Q-TOF mass spectrometer (Impact II, Bruker Daltonik GmbH; Bremen, Germany) using a Captive Spray source, interfaced with a nano-HPLC RSLC System (Ultimate 3000 Thermo Scientific). Samples were concentrated on a pre-column (Thermo Scientific, C18 PepMap100, 2 cm × 100 μm ID, 5 μm particle size, 100 A) at a flow rate of 10 μL/min using 0.1% tri-fluoro acetic acid. After pre-concentration, peptides were separated on a Thermo Scientific C18 PepMap100 UHPLC column (50 cm x 75 μm ID, 2 μm particles, 100A) at a flow rate of 400 nL/ min using a 90 min linear gradient (buffer A: 2% ACN in 0.1% FA; buffer B 100% ACN in 0.1% FA). Acquisition is data dependent InstantExpertise™ mode (Bruker Daltonik GmbH; Bremen, Germany) for precursor selection based on fixed time (1 MS every 2 sec at 0.25 sec/scan) for MS and as many as possible MSMS in between (1.75 sec) at acquisition time from 0.0625s to 0.25 s/scan based on precursor intensity.