In NPrEC, we identified 116 protein-SNOs, with modification sites located for 86 of them by the use of a combined protocol involving both protein and peptide pull-down following the BST. Of the proteins identified, 60% belong to four functional categories: cell structure/cell motility/intracellular protein trafficking, protein folding/stress response/protein assembly, RNA splicing/processing/transcription regulation, and metabolisms. Thirty percent of the proteins identified have previously been reported to be nitrosylated in non-prostate tissues, and certain proteins have been shown to cause aberrant signaling processes and/or been associated with disease development. For example, several isomerases (triosephosphate isomerase 1 TIP1; glucose phosphate isomerase GPI, protein disulfide isomerase family A, member 3 and 6, PDI, proline 4-hydroxyase, P4HB) and oxidative stress proteins (e.g., peroxiredoxin) were identified as nitrosylated, among which SNO-PDI 
and SNO-peroxiredoxin 
have been reported to serve as direct links between nitrosative/oxidative stress and neurodegenerative diseases. Although NS has been implicated in the pathogenesis of prostatic diseases 
, its mechanism of action has not been established. The discovery of these protein-SNOs in this study support the notion that, depending on the level of NS, NO can induce modification on a specific subset of proteins that may play a role in the pathogenesis of prostate diseases.
A number of recent studies have described similar peptide pull-down approaches for the profiling of nitrocysteine-containing peptides in cells or lysates treated with either CysNO or nitrosoglutathione (GSNO) 
. Compared with these studies, which identified nitrosylated targets on the basis of one single biotinylated peptide, our study strengthened the stringency/confidence of the peptide-SNO identifications by also identifying the corresponding proteins in the protein pull-down. In fact, by taking advantage of the high mass accuracy measurement of the LTQ-Orbitrap, in our study, all the sequenced biotinylated peptides (Table S1A and S1B
) have a low precursor mass error <5 ppm (although a peptide mass tolerance was set to <15 ppm during database searching), strongly supporting our peptide-SNO identifications. Of the sites of modification we identified in the study, 16% have been reported in the literature. These sites, together with the newly located sites, serve as a foundation for future investigations of how NO regulates the functions of the target proteins.
In our analysis of the 141 SNO sites, we did not find any consensus sequence motif in terms of primary or secondary structures. This is consistent with the previously reported results of primary sequence analysis of SNO proteins, including an attempt to derive predictive signatures by using machine-learning approaches by Hao and colleagues 
. On the other hand, hydrophobicity analysis indicates that half of the SNO sites are located within a hydrophobic pocket, which is also in agreement with the findings of Greco and colleagues 
, who found that most of the 18 SNO sites they analyzed were located in hydrophobic pockets. We observed an enrichment of buried cysteine residues (79%) in SNO proteins, which may be indicative of the primary role of S-
nitrosylation in regulating protein stability. In fact, many studies have demonstrated that nitrosylation can affect protein stability (e.g., HIF-1α 
, Bcl-2 
)—some through the ubiquitin-proteasome system. For the SNO sites that can be mapped to structurally resolved structures, half of the buried cysteines were surrounded by spatially adjacent charged amino acids. Perez-Mato et al. first proposed an autocatalytic mechanism of nitrosylation that is facilitated by the charged amino acids in the vicinity of the cysteine thiol 
. While this phenomenon has been demonstrated in a few proteins (e.g., MAT, 14-3-3θ, CLIC4) 
, our data, with its larger collection of structures, further enhances the idea of an autocatalytic mechanism, at least in half of the cases. Finally, analysis on the mapped structures revealed that nitrosylation might affect the specific function of some proteins. In the 24 mapped structures, the function of several proteins has previously been demonstrated to be affected by nitrosylation. For example, in GAPDH, nitrosylation of C152, which is in the NAD binding pocket, has been shown to abolish the catalytic activity of GAPDH by recruiting the E3-ubiquitin-ligase Siah1 
. Moreover, we also identified some new SNO sites, including those in EGFR and PCNA. The kinase activity of EGFR has previously been shown to be affected by NO 
. Structural analysis revealed that one of the SNO sites is involved in the ligand-binding surface of EGFR, and nitrosylation of this site may affect its ligand binding. For PCNA, since C81 is located at the trimer interface of PCNA, nitrosylation of this site may affect the trimerization/stability of the trimer ring formation. Although functional assays are necessary to confirm these hypotheses, mapping of SNOs into structures represents a strategy for predicting functional SNOs.
Considering that several groups have demonstrated the effects of NO on cell proliferation, migration, motility, adhesion, and aggressiveness in PCa cells 
, we decided to focus on a subset of protein-SNOs we identified that are related to cancer initiation and progression. We confirmed, by western-blot analysis, a total of six representative proteins, including the proliferating cell nuclear antigen (PCNA), karyopherin-β, α-catenin, integrin β4, maspin, and eEF1A1. PCNA is critical during chromosomal DNA replication; it has been found to be overexpressed in various types of cancers and that its expression is associated with poor survival outcomes 
. Karyopherin-β1 is instrumental in nucleo-cytoplasmic transport of signaling molecules. It is overexpressed in cervical cancer 
, and its down-regulation impairs cell proliferation 
. Integrin β4 is a transmembrane protein expressed predominantly on hemidesmosomes of epithelial cells. Similar to other members of the integrin family, integrin β4 mediates anchorage and migration of normal and cancer cells via influencing cell-matrix and cell-cell interactions 
. α-Catenin forms the link between the β-catenin/E-cadherin complex and the actin cytoskeleton, hence playing a key role in maintaining cell adhesion 
. Loss of integrins and catenins has been found in primary and metastatic PCa 
. Maspin is a 42-kDa serine protease inhibitor with multifaceted tumor suppressive activities in breast, prostate, colon, and oral squamous cancers. Of interest is its biphasic expression pattern during carcinogenesis, with a loss of expression during early steps of tumorigenicity and re-expression in metastatic cancer 
. Maspin-transfected PCa cells exhibit reduced tumorigenicity, vasculature, and metastatic potential under hypoxic conditions 
. The eEF1 subunit 1A1 is involved in the binding of tRNA to ribosomes during protein synthesis. It has been found to be overexpressed in PCa 
and implicated in tumorigenesis, signal transduction, and apoptosis 
. While the effect of NO in the pathogenesis of BPH and PCa is unclear, our findings are concordant with the fact that signaling molecules important in cancer development are targets of nitrosylation and therefore may be involved in NS-
induced initiation of the NPrEC. Although a number of apoptotic or cell cycle–related proteins whose activity or stability can be modulated by S-
, SNO-Bcl-2 was shown to promote the malignant transformation of lung epithelial cells 
, supporting the role of NS in tumorigenesis 
. It is conceivable that the previously reported over-expression of iNOS 
, together with other oxidative insults 
, in the prostate epithelial and stromal compartments is likely to cause nitrosylation of the above mentioned protein targets, leading to modulation of cell growth, disruption of cellular architecture, and/or transformation of normal epithelial cells of the prostate.
Over-expression of NOS has been shown to promote tumorigenicity in other cancers, and the therapeutic application of NOS inhibitors for chemopreventive purposes has been the subject of intense research for the past decade 
. Inhibition of NOS was recently shown to have tumor antivascular activity in patients with PCa 
, and the efficacy of NO-releasing drugs for BPH and lower urinary tract symptomatology was evaluated in a clinical trial based on the rationale that NO can relax muscular tone 
. Given that the emerging evidence suggesting the importance of NS in carcinogenesis and inflammation-related diseases 
, further elucidation of the functional significance of the targets identified in the present study should not only yield insights into details of the complex role of NO and inflammation-related prostatic diseases but also provide the experimental basis for NOS-related therapeutics.