Symmetry c18 trap column

The resulting peptides were analyzed using a nanoAcquity liquid chromatography (Waters) coupled with a Q Exactive HF-X (Thermo fisher scientific). Samples were analyzed randomly, loaded on a Symmetry C18 trap column (20 mm × 0.18 mm, 5 µm, Waters), and resolved on an HSS T3 (250 mm × 0.075 mm, 1.8 um, Waters) analytical column at 350 nl/min, using a gradient of 4–27%B (MeCN, 0.1% formic acid) for 155 min. MS1 acquisition was performed at m/z range of 375–1650 m/z at 120,000 resolution (@400 m/z), allowing Automatic Gain Control (AGC) target of 10⁶ with a maximum Injection Time (IT) of 60 ms. MS2 acquisition was performed on the Top10 ions at Data-Dependent Acquisition (DDA) using Higher-energy Collisional Dissociation (HCD) fragmentation set at 27 Normalized Collision Energy (NCE) acquired at 15,000 resolution (@200 m/z). IT was set to 60 ms and AGC to 1e5. Dynamic exclusion was set to 30 s with a counter of 1. The resulting data was processed with MaxQuant (v1.6.6.0). The data were searched with the Andromeda search engine against the Human proteome database (SwissProt Nov20) appended with common lab protein contaminants. The following modifications were allowed: fixed carbamidomethylation on C, variable protein N-terminal acetylation, variable deamidation on NQ and variable oxidation on M. The quantification was based on the LFQ method, based on unique peptides.

The purified proteins were analyzed by LC-MS/MS. The proteins were trypsin digested by a standard in-gel digestion protocol, and analyzed by LC-MS/MS on an LTQ Orbitrap XL (Thermo Scientific) equipped with a nanoACQUITY UPLC system (Waters). A Symmetry C18 trap column (180 μm x 20 mm; Waters) and a nanoACQUITY UPLC column (1.7 μm, 100 μm x 250 mm, 35°C) were used for peptide separation. Trapping was done at 15 μl min⁻¹, 99% buffer A (water with formic acid (0.1%)) for 1 min. Peptide separation was performed at 300 nl min⁻¹ with buffer A and buffer B (CH₃CN containing 0.1% formic acid). The linear gradient was from 5% buffer B to 50% B at 50 min, to 85% B at 51 min. MS data were acquired in the Orbitrap with one microscan, and a maximum inject time of 900 ms followed by data-dependent MS/MS acquisitions in the ion trap (through collision induced dissociation, CID). The Mascot search algorithm was used to search for the appropriate noncanonical substitution (Matrix Science, Boston, MA).

The predominant bands at ∼23 kDa and 38 kDa from a Coomassie-stained gel were excised and sent to the Keck MS Proteomics Resource at Yale University School of Medicine where they were digested and analyzed to identify peptides and their corresponding proteins. Briefly, the gel slices were first washed with 50% acetonitrile/50% water followed by 50 mM ammonium bicarbonate/50% acetonitrile/50% water. It was then dried, rehydrated with trypsin in 10 mM ammonium bicarbonate, and digested at 37 ^°C for 16 h. The resultant peptides were separated using a Waters Symmetry C18 trap column and a nanoAcquity UPLC separating column.Elution was performed with a 52-min linear gradient of 5 to 50% acetonitrile in 0.05% formic acid. The LC MS/MS mass spectral data obtained from an LTQ-Orbitrap Elite mass spectrometer system were searched for analogy to known peptides/protein using the Mascot algorithm (version 2.20) for uninterpreted MS/MS spectra and searching the NCBInr database. The significance score relied on a probability-based protein/peptide identification score through searching known databases with a peptide score cutoff of 95% confidence in identification (Fig. S1, A and B). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (77 (link)) partner repository with the dataset identifier PXD027875.

Protein identification was conducted in the same way as described in [8 ]. Measurements were performed using a nanoAcquity UltraPerformance LC system connected to an auto-sampler equipped with a HSS T3 analytical column (1.8-µm particle, 75 µm × 150 mm) kept at 45 °C, and a Symmetry C18 trap column (5-µm particle, 180 µm × 20 mm), all Waters, USA. This setup was connected to an LTQ Orbitrap Elite. A 180-min gradient was used: (0–5 min: 99% buffer A and 1% buffer B, 5–10 min 99–94% A, 10–161 min: 94–60% A, 161–161.5 min: 60–14% A, 161.5–166.5 min: 14–4% A, 166.5–167.1 min: 99% A, 167.1–180 min: 99% A). To optimize the method for small cell numbers, the MS/MS max ion inject time was increased to 400 ms.

Samples were analyzed as previously described using an Acquity M-class UPLC system (Waters, Milford, MA, USA) coupled to a Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with a Nanospray Flex source operated positive polarity mode [56 (link)]. Injections (4 μL) were made to a Symmetry C18 trap column (100 Å, 5 μm, 180 μm × 20 mm; Waters) and then separated on a HSS T3 C18 column (100 Å, 1.8 μm, 75 μm × 250 mm; Waters) resulting in an average peak width of 30 s. Data was acquired using a top 20 data-dependent acquisition mode with an isolation window of 1.5 m/z. Survey scans were collected with a scan range of 350–2000 m/z, 120,000 resolving power, an AGC target of 1 × 10⁶, and maximum injection time of 50 ms. Precursor ions were selected (isolation window of 1.5 m/z) for higher-energy collisional dissociation (HCD) collecting spectra with a scan range of 200–2000 m/z, resolving power of 30,000, AGC target of 3 × 105 and a maximum injection time of 100 ms. The total duty cycle of this method is 2.05 s producing approximately 15 survey scans across a chromatographic peak.