Eksigent nanolc 400 system

Nitroso dapsone-modified peptides were characterized using a TripleTOF 6600 (AB Sciex) mass spectrometer. Briefly, samples were delivered into the mass spectrometer by automated in-line reversed phase liquid chromatography, using an Eksigent NanoLC 400 System (AB Sciex) mounted with a trap and analytical column (15 cm X 75 μm). A NanoSpray III source was fitted with a 10 μm inner diameter PicoTip emitter (New Objective). A gradient of 2–50% (v/v) acetonitrile/0.1% (v/v) formic acid over 90 min was applied to the column at a flow rate of 300 nl/min. Spectra were acquired automatically in positive ion mode using information dependent acquisition, using mass ranges of 400–1600 Da in MS and 100–1400 Da in MS/MS. Up to 25 MS/MS spectra were acquired per cycle (approximately 10 Hz) using a threshold of 100 counts per s, with dynamic exclusion for 12 s and rolling collision energy.

Concentration of serum proteins was measured and 100 μg protein samples were prepared for mass analysis. Proteins were reduced by treatment with 5 mM Tris (2-carboxyethyl) phosphine (Pierce, Rockford, IL, USA) at 37 °C, 300 rpm, for 30 min. For alkylation, 15 mM Iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA) was added to the samples at 25 °C, with agitation at 300 rpm for 1 h in the dark. Proteins were cleaved into peptides, using trypsin, overnight at 37 °C. Mass spectrometry-grade trypsin gold (Promega, Madison, WI, USA) was used for the purpose. After a sample containing chemical reagents was cleaned by a C18 cartridge (Waters, Milford, MA, USA), it was separated based on isoelectric point using the OFFGEL Fractionator with a 12-well setup (3100 OFFGEL Low Res Kit, pH 3–10; Agilent Technologies, Santa Clara, CA, USA). To identify the proteins, the peptide fraction that was separated into 12 samples was analyzed using a TripleTOF 5600 mass spectrometer (AB SCIEX, Concord, ON, Canada) combined with an Eksigent nanoLC 400 system and cHiPLC^® (AB SCIEX, Concord, ON, Canada).

A 12-well fractionator (3100 OFFGEL Low Red Kit, pH 3–10; Agilent Technologies) was used to separate proteins according to their isoelectric points, following the manufacturer’s protocol. This method was used to identify as many peptides as possible while preparing the library. A TripleTOF 5600 mass spectrometer (AB Sciex, Concord, Ontario, Canada) coupled with an Eksigent NanoLC 400 system and cHiPLC^®® spectrometer (AB Sciex) was used for IDA analysis. In each run, 2 µL sample solution (0.5 µg/µL) was loaded onto an Ekigent ChromXP NanoLC trap column (350 µm i.d. × 0.5 mm, ChromXP C18 3 µm) at a flow rate of 5000 nL/min for 5 min. The injected sample was eluted from the Eksigent Chrom XP NanoLC column (75 µm i.d. × 15 cm), at a flow rate of 300 nL/min for 120 min with a gradient of 5–90% mobile phase B. The following LC gradient was used: (time/mobile phases B%) 0 min/5%, 10.5 min/40%, 111.5 min/90%, 112 min/5%, and 120 min/5%. Mobile phase B was 90% acetonitrile/0.1% formic acid in HPLC-grade water, and mobile phase A was 0.1% formic acid in HPLC-grade water. The mass-to-charge ratio (m/z) MS scan range was m/z 250–2500, and MS/MS scan range was m/z 100–2500.