Acclaim pepmap 100 trap column

LC–MS/MS analysis was carried out using the Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) coupled to the Easy nLC1200 nano-flow UPLC. Peptides (500 ng of each sample) were injected into the Acclaim PepMap 100 trap column (nanoViper C18, 100 μm × 2 cm, Thermo Scientific) and separated by the Acclaim Pep Map RSLC analytical column (nanoViper C18, 50 μm × 15 cm, Thermo Scientific) set at the flow rate of 300 nL/min. The solvent gradients were set as a linear gradient of 8~38% mobile phase B (80% acetonitrile contains 0.1% formic acid) over 102 min through 120 min run time. The sample was atomized using the nanoESI source. The data-dependent acquisition (Top 20) was carried out using MS survey scans in the 350~1700 m/z range with 70,000 mass resolution. For subsequent MS/MS analysis, the resolution was set to 17,500, and the isolation window was set to 1.6 m/z. The normalized collision energy was 27 eV. Peptides with charge 2~7 were selected.

Extracted peptides were separated by a Ultimate 3000 RSCLnano System (Thermo Fisher Scientific) with a Acclaim PepMap100 trap column (3 µm C18 particle size, 100 Å pore size, 75 µm inner diameter, 2 cm length, Thermo Fisher Scientific) as a precolumn using 0.1% TFA as a mobile phase and a Acclaim PepMapRSLC (2 µm C18 particle size, 100 Å pore size, 75 µm inner diameter, 25 cm length, Thermo Fisher Scientific) as analytical column. The flow rate was constant with 300 nl/min using a 2 h gradient of 0.1%FA (Fluka) to 0.1% FA/60% acetonitrile. Separated peptides were eluted via nano electrospray ionization into the mass spectrometer (QExactive hybrid quadrupole-orbitrap mass spectrometer, Thermo Fisher Scientific). Mass spectra were recorded in positive ion mode with a mass range of 300–2,000 m/z and a resolution of 70,000. Up to ten precursors (+2, +3 charge states) were isolated within a 2 m/z isolation window and fragmented via higher-energy collisional dissociation. MS/MS spectra were recorded in centroid mode with a maximal ion time of 60 ms and a target value for the automatic gain control set to 100,000. The resolution was 17,500 at a scan range of 200 to 2,000 m/z. Already fragmented precursors were excluded from further isolation for the next 100 s.

The proteomic analysis was carried out at Monash Biomedical Proteomics Facility, Monash, Australia. Data-dependent acquisition was performed on a QExactive™ Plus 1 mass spectrometer (Thermo Scientific, Waltham, MA, USA) coupled to a Dionex UltiMate^® 3000 RSLC nano Liquid Chromatography (LC) system (Thermo Scientific, Waltham, MA, USA) equipped with Acclaim PepMap RSLC analytical column (75 µm × 50 cm, nanoViper, C18, 2 µm, 100Å, Thermo Scientific, Waltham, MA, USA) and Acclaim PepMap 100 trap column (100 µm × 2 cm, nanoViper, C18, 5 µm, 100Å, Thermo Scientific, Waltham, MA, USA).

Samples were analyzed on an EASY-nLC1000 system (ThermoFisher Scientific) coupled to an LTQ-Orbitrap Velos pro (ThermoFisher Scientific, Sunnyvale, CA, USA). Peptides for analysis were loaded via an Acclaim PepMap 100 trap column (100 µm × 2 cm, nanoViper, C18, 5 µm, 100 Å, Thermo Scientific), and subsequent peptide separation was performed on an Acclaim PepMap EASY-Spray analytical column (75 µm × 15 cm, nanoViper, C18, 2 µm, 100 Å, Thermo Scientific). For each liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, peptides were loaded on a precolumn with microliter pickup. Peptides were separated at a flow rate of 250 nL/min using mobile phase acetonitrile with the addition of 0.1% formic acid in a gradient of 5–40% over 120 min. Eluted peptides were examined with an Orbitrap mass spectrometer using a spray voltage of +1.8 kV. A full scan from m/z 300 to 2000 at a resolution of 60,000 was acquired. Top N mode followed by a ten data-dependent acquisition of MS/MS scan method was applied using CID with normalized energy of 35 eV. Dynamic exclusion for previously fragmented precursor ions was used with the following parameters: exclusion time 180 s, repeat count 1, repeat duration 30 s, exclusion mass width 10 ppm, exclusion size 500 m, signal threshold 5000, and isolation width 1 m/z. Singly charged species were excluded from fragmentation.

The peptide mixture was loaded onto the Thermo EASY-nLC System equipped with Acclaim PepMap100 trap column (100 μm × 2 cm, nanoViper C18, Thermo Fisher Scientific, Shanghai, China). Peptides were separated on analytical EASY column (75 μm × 10 cm, 3 μm, Thermo Scientific) over 60 min at a flow rate of 300 nL/min consisting of buffer A (0.1% formic acid) and buffer B (84% v/v acetonitrile and 0.1% v/v formic acid). The liquid-phase linear-gradient program was as follows: 0–35% buffer B for 50 min, 35–100% buffer B for 5 min, and hold in 100% buffer B for 5 min.
MS data were acquired using Q Exactive MS (Thermo Scientific) in the positive ion mode over 300–1800 m/z at a resolution of 70,000 at m/z 200. The automatic gain control (AGC) target was set to 3 × 10⁶, and the maximum inject time to 10 ms. Precursor ions for higher-energy collisional dissociation (HCD) fragmentation were dynamically selected according to a data-dependent top 10 method. Values for MS/MS analysis were set as follows: resolution for HCD spectra was 17,500 at m/z 200, isolation width was 2 m/z, normalized collision energy was 30 eV, dynamic exclusion duration was 40 s, and underfill ratio was defined as 0.1%.

LC–MS/MS analysis were performed by NICEM (Seoul National University, Republic of Korea). A Thermo Scientific Quadrupole-Orbitrap instrument (Thermo Scientific) equipped with Dionex U 3000 RSLCnano HPLC system was used. Mass spectrometric analyses were performed using a Thermo Scientific Orbitrap Exploris 240 mass spectrometer. Fractions were reconstituted in solvent A (Water/Acetonitrile (98:2 v/v), 0.1% Formic acid) and then injected into LC-nano ESI–MS/MS system. Samples were first trapped on a Acclaim PepMap 100 trap column (100 μm x2cm, nanoViper C18, 5 μm, 100Å, Thermo Scientific, part number 164564) and washed for 6 min with 98% solvent A (water/ACN (98:2 v/v), 0.1% Formic acid at a flow rate of 4 μL/min, and then separated on a PepMap RSLC C18 column (75 μm x 15 cm, nanoViper C18, 3 μm, 100Å, Thermo Scientific, part number ES900) at a flow rate of 300 nL/min. The LC gradient was run at 2% to 8% solvent B over 10 min, then from 8 to 30% over 55 min, followed by 90% solvent B (100% ACN and 0.1% Formic acid) for 4 min, and finally 2% solvent B for 20 min. Xcaliber software version 4.4 was used to collect MS data. The Orbitrap analyzer scanned precursor ions with a mass range of 350–1800 m/z with 60,000 resolution at m/z 200. Mass data are acquired automatically using proteome discoverer 2.5 (Thermo Scientific).