Nanohplc

For identifying individual ASYN nitration sites, reversed phase liquid chromatography nanospray tandem mass spectrometry (LC-MS/MS), comprised of a Linear Trap Quadropole (LTQ) Orbitrap mass spectrometer (Thermo Fisher) and an Eksigent nano HPLC were applied. The dimensions of the reversed phase LC column were: 5 μm particle size, 100 Å pore size in a 10 cm silica capillary with an inner diameter of 75 μm (Acclaim™ PepMap™ 100C18-LC-column, Thermo Scientific). After sample injection, the column was washed for 5 min with 100% mobile phase A (0.1% formic acid) and peptides were eluted using a linear gradient of 10% mobile phase B to 40% mobile phase B within 35 min, then to 80% B in an additional 5 min, at 300 nl/min. The LTQ-Orbitrap mass spectrometer was operated in a data dependent mode in which each full MS scan (30 000 resolving power) was followed by five MS/MS scans where the five most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35% in the LTQ ion trap. Dynamic exclusion was allowed. Tandem mass spectra were analyzed by their comparison with protein databases using Mascot (Matrix Science).

The peptides (~ 3 μg per run) were suspended in 0.1% formic acid and fractionated in a reverse phase capillary column (10 cm × 75 μm, packed with 5 μm of C18 Aqua resin-Phenomenex) coupled to a nanoHPLC (Thermo) inline to a LTQ-Orbitrap Velos (Thermo Scientific). Fractionation was performed under a 60-min gradient: 0–28% buffer B (0.1% formic acid in acetonitrile) in 45 min, 28–80% in 13 min; 80–5% in 2 min at a flow rate of 300 nL min−1. The MS voltage was 1.9 kV, and the capillary temperature was set at 200 °C. The equipment was operated in data independent acquisition (DIA) with 50 m/z to select the precursor ions for fragmentation as described in [86 ]. For each cycle, one full MS1 was followed by 8 MS2 scans using an isolation width of 50 m/z starting from 325 to 1075 m/z.

Lyz_red (1.25 μM) was incubated with GSSG (0.4 mM) in 0.01 M potassium phosphate buffer, pH 7.4. The reaction was stopped after 10 seconds or 10 min by adding 0.25 mM bromopyruvate which alkylated residual protein cysteines within 1–2 sec. Then the samples were lyophilized. A Lyz_red solution (1.25 μM) was immediately alkylated with bromopyruvate and used as control. Samples were resuspended in 0.2% TFA and desalted by reversed-phase HPLC on a Phenomenex Jupiter C4 column (250 mm × 2.0 mm, 300 Å pore size) with a linear gradient from 10% to 95% of solvent B (0.07% TFA in 95% acetonitrile) in 30 min, at a flow rate of 200 μL/min using an Agilent Technologies 1100 HPLC (Agilent Technologies, USA). Protein fractions were collected and lyophilized. Controlled pepsin hydrolysis was carried out by dissolving the samples in 5% formic acid, pH 2.5 and adding pepsin at an enzyme to substrate ratio of 1:300 w/w at 37 °C for 2 hours. Sample was then lyophilized, resuspended in 0.2% formic acid and directly analyzed by nanoLC/MS-MS on an LTQ-XL Orbitrap mass spectrometer equipped with a nanoHPLC (ThermoFisher, USA). Peptides containing modified cysteine residues were selected using the ion extraction chromatograms of the corresponding doubly and triply charged ions and the assignments were confirmed by manual inspection of their fragmentation spectra.