**2.4. Identification of nitroproteins with MS/MS**

proteins in a human nonfunctional pituitary adenoma tissue [11], 18 nitroproteins and their 23 nitrotyrosine sites in human astrocytoma tissues [35], were identified with 2DE-MS/MS. The nitration site was located onto the corresponding functional domain, and each nitroprotein

A clinically nonfunctional human pituitary adenoma tissue was obtained during surgery. The expression of FSH, LH, GH, prolactin, TSH, and ACTH were all negative in tumor cells [11]. A normal human pituitary tissue acted as control group was obtained from the post-mortem sample (a drowning male) [9, 36]. Human astrocytoma brain tissues (including different clinical staging-I/II/III/IV) were obtained from the Department of Neurosurgery of Xiangya Hospital, China [35]. The tissues were frozen in liquid nitrogen immediately, then stored at −80°C. According to the protein extraction manuals (Pierce, Rockford, IL, USA),supernatant

2DGE: The precast IPG strips (pH 3–10 NL; 180 × 3 × 0.5 mm) and 18-cm IPGstrip holder was used for 2DGE first dimension—isoelectic focusing (IEF) on an IPGphor instrument (GE Heathcare) to separate protein sample. After IEF, the IPG strip was processed for 2DGE second dimension—sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), namely, the equilibrated proteins in the IPG strips were separated by molecular weight during electrophoresis on in 12% PAGE resolving gel (250 × 215 × 1.0 mm), and visualized with

2DGE-based Western blotting: The 2DGE-separated proteins were transferred to a PVDF membrane, which were blocked by BSA, incubated with an anti-human nitrotyrosine antibody and secondary antibody, then visualized with 1-Step™ nitro blue tetrazolium/5-bromo-

Image analysis of a 2D gel and of 2D-Western blotting: The scanned images of the 2D gels and the Western blot membranes were input to a PDQuest analysis system (Bio-Rad, version 7.1, Hercules, CA) to synthesize image. Gaussian spots were applied to all subsequent spotmatching and analyses. Each spot volume was normalized to the total optical density (OD) to

LC-ESI-MS/MS: The nitrotyrosine-positive gel-spots were excised, digested, purified, eluted, air-dried, redissolved. Then, the peptide mixture was subjected to LC-ESI-quadrupole-time of flight (LC-ESI-qTOF) or LTQ-OrbiTrap Velos MS/MS analyses. The detailed operation process

was carried out pathway analysis to speculate the possible biological function.

**2. Materials and methods**

110 Electrophoresis - Life Sciences Practical Applications

**2.1. Tissues and extraction of proteins**

of the tissue lysate was extracted for further analysis.

**2.2. 2DGE-based western blot detection of nitroproteins**

silver-staining [9, 36] or Coomassie brilliant blue G staining [35].

minimize experimental factors on a spot volume [30].

and parameter settings have been described [35].

4-chloro-3-indolyl phosphate (NBT/BCIP) (Thermo Product No. 3404).

**2.3. MS/MS characterization of tryptic nitropeptides from nitroproteins**

MS/MS data were used to identify the protein and nitrotyrosine sites by searching the SwissProt and NCBInr databases with SQUEST or Mascot software, with mass modifications of +45 Da (+NO<sup>2</sup> – H) at Tyr, +57 Da (+NH<sup>2</sup> COCH<sup>2</sup> – H) at Cys, +16 Da (oxidation) at Met. Protein domains and motifs analyses were carried out with ScanProsite software (http:// us.expasy.org/tools/scanprosite). Nitrotyrosine sites within a given domain or motif were determined with MotifScan software (http://myhits.isb-sib.ch/cgi-bin/motif\_scan). The functions and experimental data-based model of nitroproteins was searched on the Swiss-Prot database annotation page and the related literature resources.
