C18 trap column

DDA (data dependent acquisition) fractions and Data-dependent acquisition (DDA) samples were performed on a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled with an Ultimate 3,000 RSLCnano system (Thermo Fisher Scientific). A nano-LC column (150 μm × 30 cm, 1.8 μm, 100 Å) was packed in-house for peptide separation at a flow rate of 500 nL/min. For DDA analysis, peptides were loaded onto a C18 trap column (300 μm × 5 mm, 5 μm, Thermo Scientific) with buffer A (2% ACN, 0.1% FA) for 5 min, then it was eluted with a gradient from 5 to 25% buffer B (98% ACN, 0.1% FA) for 155 min, 25–30% buffer B for 10 min, and 30–80% buffer B for 5 min. The mass spectrometry parameters were set as follows: MS scan range 350–1,500 m/z; loop count 30; NCE 28; MS resolution 120,000, maximal injection time (MIT) 50 ms; MS/MS HCD scans with resolution 30,000, MIT 100 ms; dynamic exclusion duration 30 s; isolation window 2.0 m/z; intensity threshold 2.0 e4; charge exclusion, excluding 1, 7, 8, >8. For DIA (data independent acquisition) analysis, the same nano-LC system and gradient were for used as DDA analysis. The DIA MS parameters were set as follows: full scan range 400–1250 m/z at resolution 120,000 with MIT 50 ms; DIA isolation window was set to 17 m/z with loop count 50 and automatic MIT, scanned at resolution 30,000; stepped NCE: 22.5, 25, and 27.5; AGC target 1e6.

Proteomics sample preparation and analysis were performed as previously described.^{148 (link)} Briefly, protein precipitate was sonicated and reduced using DTT before being digested by sequence-grade trypsin and desalted. Peptides were TMT labelled using the TMT 10-plex isobaric label reagents (Thermofisher) and fractionated on a Waters XBridge BEH C18 (3.5 μm, 4.6 × 250 mm) reverse phase column using an Agilent 1100 HPLC system. Phosphopeptides were enriched using titanium dioxide coated magnetic beads. Fractionated samples and bead-enriched samples were individually loaded onto a C18 trap column (3 μm, 75μm × 2 cm, Thermo Fisher Scientific) connected in-line to a C18 analytical column (2 μm, 75 μm × 50 cm, Thermo EasySpray) using the Thermo EasyLC 1200 system. Peptides were ionized using a nanospray ion source into a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). MS/MS spectra were searched against the Uniprot human complete proteome FASTA database downloaded on 2018_10_26, using the MaxQuant software (Version 1.6.7.0) that integrates the Andromeda search engine.

LC-MS/MS data acquisition was carried out on a Q Exactive plus mass spectrometer coupled with an Easy-nLC 1200 system (both Thermo Scientific). Peptides were first loaded onto a C18 trap column (75 μm × 2 cm, 3 µm particle size, 100 Å pore size, Thermo) and then separated in a C18 analytical column (75 μm × 250 mm, 2 µm particle size, 100 Å pore size, Thermo). Mobile phase A (0.1% formic acid) and mobile phase B (80% ACN, 0.1% formic acid) were used to establish the separation gradient. A constant flow rate was set at 300 nL/min. For DDA mode analysis of TMT samples, each scan cycle is consisted of one full-scan mass spectrum (R = 70 K, AGC = 3e6, max IT = 50 ms, scan range = 350–1800 m/z) followed by 15 MS/MS events (R = 35 K, AGC = 1e5, max IT = 50 ms). HCD collision energy was set to 32. Isolation window for precursor selection was set to 1.2 Da. Former target ion exclusion was set for 45 s.

The samples were subjected to treatment with ultra-sonication, and proteins concentration was determined by BCA kit. After peptide purification using SDB-RPS desalting column, all samples were analyzed on an UltiMate 3000 RSLCnano system coupled on-line with Q Exactive HF mass spectrometer through a Nanospray Flex ion source (Thermo) following user manual. Peptide samples were injected into a C18 Trap column (75 µm*2 cm, 3 µm particle size, 100 Å pore size, Thermo), and seperated in a reversed-phase C18 analytical column packed in-house with ReproSil-Pur C18-AQ resin (75 µm*25 cm, 1.9 µm particle size, 100 Å pore size). The MS was operated in DDA top20 mode with a full scan range of 350–1500 m/z. MS raw data were analyzed with MaxQuant (V1.6.6.0) using the Andromeda database search algorithm. Proteins with a fold change > 1.5 or < 1/1.5 and a students’ t-tests P-value < 0.05 were identified as differentially expressed proteins (DEPs) and used for functional annotation.

Each sample was first separated using the nanoliter flow HPLC system Easy nLC1000 (Sigma, Japan). After equilibrating the column with 95% buffer A (0.1% FA (Sigma, Japan)), samples were loaded from the autosampler to the C18 trap column (3 μm, 0.10 μm × 20 mm) (Thermo Scientific, USA) and separated by an analytical C18 column (1.9 μm, 0.15 μm × 120 mm) (Thermo Scientific, USA) at a flow rate of 600 nL/min. Primary parent ion scanning was performed in the orbitrap after peptide fragmentation. The scanning range was set to 300 ~ 1,400 (m/z), the Orbitrap resolution (Thermo Scientific, USA) was set to 120,000, the automatic gain control target was set to 5 × 10⁵, and the maximum injection time was set to 50 ms. HCD activation type was used in secondary spectrograms (ddMSnScan) with a Iontrap, a collision energy was 30% and the stepped collision energy was 5%. The dynamic exclusion time was set to 18 s. The spray voltages of the positive and negative ion mode mass spectrometers were set to 2,000 V and 600 V separately, and the spray temperature was 320 ℃ for peptides.

Samples consisted of approximately 1 μM of in-solution digested product. For each proteolytic, enzyme digestion liquid chromatography was performed on a Thermo nLC1200 (Thermo Fisher Scientific Inc., Waltham, MA, USA) in single-pump trapping mode with a Thermo PepMap RSLC C18 EASY-spray column (2 μm, 100 Å, 75 μm × 25 cm) and a Pepmap C18 trap column (3 μm, 100 Å, 75 μm × 20 mm). The solvents used were A: water with 0.1% formic acid and B: 80% acetonitrile with 0.1% formic acid. Samples were separated at 300 nL/min with a 250 min gradient starting at 3% B increasing to 30% B from 1 to 231 min, then to 85% B at 241 min, holding for 10 min.
Mass spectrometry data were acquired on a Thermo Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) in data-dependent mode. A full scan was conducted using 60 k resolution in the Orbitrap in positive mode. Precursors for MS² were filtered by monoisotopic peak determination for peptides, intensity threshold 5.0 × 10³, charge state 2–7, and 60 s dynamic exclusion after 1 analysis with a mass tolerance of 10 ppm. Higher-energy C-trap dissociation (HCD) spectra were collected in ion trap MS² at 35% energy and isolation window 1.6 m/z.

Liquid chromatography was performed on a Thermo Scientific Ultimate 3000 RSLC [23 , 24 ] equipped with a 20 mm × 100 µm C₁₈ trap column (Thermo Scientific) and a CSH 25cmx75µm 1.7 µm particle size C₁₈ column (Waters) analytical column. The solvent system employed was loading: 2% acetonitrile:water; 0.1% FA; Solvent A: water; 0.1% FA and Solvent B: 100% acetonitrile, 0.15% FA. Methods were previously described in [1 (link)]. Separation was effected using a linear gradient from 2 B to 30% B over 65 min followed by 30 B to 45% B from 65 to 80 min. The sample was loaded onto a trap column at 2 µL/min before switching to the analytical column at three minutes with a flow rate of 300 nL/min. Chromatography was performed at 45 °C and the outflow delivered to the mass spectrometer through a stainless-steel nano-bore emitter.