Chromxp

Tryptic peptides of plasma samples were separated on a NanoLC 425 System (SCIEX). The flow was 5 μl/min with trap-elute setting using a 0.5 × 10 mm ChromXP (SCIEX). The LC gradient was set to a 43-min gradient from 4–32% B with 1 h total run. Mobile phase A was 100% water with 0.1% formic acid. Mobile phase B was 100% acetonitrile with 0.1% formic acid. We used an 8-μg load of undepleted plasma on a 15-cm ChromXP column. MS analysis was performed using SWATH acquisition on a TripleTOF 6600 system equipped with a DuoSpray source and 25-μm ID electrode (SCIEX). Variable Q1 window SWATH Acquisition methods (100 windows) were built in high-sensitivity MS/MS mode with Analyst TF Software 1.7.

Equal concentrations of protein samples were trypsinized, and digested proteins were analyzed using a TripleTOF 5600 MS (AB Sciex, Foster City, CA, United States) equipped with an Eksigent MicroLC 200 system (Eksigent, Dublin, CA, United States) with an Eksigent C18 reverse-phase column (150 × 0.3 mm, 3 μm, 120 Å) (Sharma et al., 2019a (link)). For protein identification, spectral libraries were generated using information-dependent acquisition (IDA) mode after injecting 2 gm of tryptic digest on the column using an Eksigent NanoLC-Ultra^TM 2D Plus system coupled with a SCIEX Triple TOF^® 5600 system fitted with a NanoSpray III source. The samples were loaded on the trap (Eksigent Chrom XP 350 μm × 0.5 mm, 3 μm, 120 Å) and washed for 30 min at 3 μl/min. A 120 min gradient in multiple steps (ranging from 5 to 50% acetonitrile in water containing 0.1% formic acid) was set up to elute the peptides from the ChromXP 3-C18 (0.075 × 150 mm, 3 μm, 120 Å) analytical column. Technical replicates of the nanoLC-TripleTOF 5600 MS experiments were performed.

Lyophilized peptide fractions were resuspended in 2% acetonitrile containing 0.1% formic acid. Aliquots of 4 μL were loaded onto a ChromXP C18 (3 μm, 150 Å) trap column and chromatographic separation performed on the Ekspert nanoLC 415 system (SCIEX, Concord, ON, Canada). The iTRAQ-labeled peptides were separated using analytical columns (ChromXP, Eksigent). High-resolution LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific). The mass spectrometry scan was set to a full scan charge-to-mass ratio m/z range of 300-1600, and the 10 highest peaks were scanned via MS/MS. All MS/MS spectra were collected in the positive ion mode using data-dependent high-energy collisional fragmentation. Raw data were processed using Proteome Discoverer 2.4 (Thermo Scientific), and searches against the UniProt protein human database were performed using ProteinPilot software (version 5.0, SCIEX). The false discovery rate (FDR) for peptides was set at 1%.

The lyophilized peptide was resuspended in 2% acetonitrile containing 0.1% formic acid, and 4-µL aliquots were loaded into a ChromXP C18 (3 μm, 120 Å) trap column. The online chromatography separation was performed on the Ekspert NanoLC 415 system (SCIEX, Concord, ON). The trapping and desalting procedures were carried out at a flow rate of 4 μL/min for 5 min with 100% solvent A (water/acetonitrile/formic acid 98/2/0.1%). Then, an elution gradient of 8-38% solvent B (water/acetonitrile/formic acid 2/98/0.1%) was used on an analytical column (75 μm × 15 cm C18-3 μm 120 Å, ChromXP, Eksigent) over 25 min. IDA (information-dependent acquisition) MS techniques were used to acquire tandem MS data on a Triple TOF 6600 tandem mass spectrometer (Sciex, Concord, Ontario, Canada) fitted with a Nanospray III ion source. Data were acquired using an ion spray voltage of 2.4 kV, curtain gas of 35 PSI, nebulizer gas of 12 PSI, and an interface heater temperature of 150 °C. The MS was operated with TOF-MS scans. For IDA, survey scans were acquired in 250 ms and up to 40 product ion scans (50 ms) were collected if a threshold of 260 cps with a charge state of 2–4 was exceeded. A rolling collision energy setting was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for 16 s.

Each labeled peptide was dissolved with 2% acetonitrile (ACN) containing 0.1% formic acid (FA). The online chromatography separation was performed on an Eksigent nanoLC 415 system (SCIEX, USA) using ChromXP C₁₈ column (3 μm, 75 μm × 15 cm, 120 A, ChromXP, Eksigent). The flow rate was 300 nL/min, and the linear gradient was 90 min (Mobile phase A: 2% ACN/0.1% FA, Mobile B: 95% ACN/0.1% FA). A Triple TOF 6600 tandem mass spectrometer (SCIEX, USA) was applied in MS analysis of the separated fractions. According to Zhu et al. [37 (link)], the data were acquired with a 2.4 kV ion spray voltage, 35 psi curtain gas, 12 psi nebulizer gas, and an interface heater temperature of 150 °C. The MS was scanned in IDA (Information-dependent acquisition) mode with a mass range 400-1,500 and an accumulation time of 250 ms. In each IDA cycle, 40 MS/MS spectra (80 ms, mass range 100-1,500) exceeding a threshold of 260 cps with a charge state of 2-4 were acquired. A rolling collision energy setting was applied to all precursor ions for collision-induced dissociation (CID), and the dynamic exclusion time was set for 16 s.