Nitroproteomics is instrumental for stratification and targeted treatments of astrocytoma patients: expert recommendations for advanced 3PM approach with improved individual outcomes

Protein tyrosine nitration is a selectively and reversible important post-translational modification, which is closely related to oxidative stress. Astrocytoma is the most common neuroepithelial tumor with heterogeneity and complexity. In the past, the diagnosis of astrocytoma was based on the histological and clinical features, and the treatment methods were nothing more than surgery-assisted radiotherapy and chemotherapy. Obviously, traditional methods short falls an effective treatment for astrocytoma. In late 2021, the World Health Organization (WHO) adopted molecular biomarkers in the comprehensive diagnosis of astrocytoma, such as IDH-mutant and DNA methylation, which enabled the risk stratification, classification, and clinical prognosis prediction of astrocytoma to be more correct. Protein tyrosine nitration is closely related to the pathogenesis of astrocytoma. We hypothesize that nitroproteome is significantly different in astrocytoma relative to controls, which leads to establishment of nitroprotein biomarkers for patient stratification, diagnostics, and prediction of disease stages and severity grade, targeted prevention in secondary care, treatment algorithms tailored to individualized patient profile in the framework of predictive, preventive, and personalized medicine (PPPM; 3P medicine). Nitroproteomics based on gel electrophoresis and tandem mass spectrometry is an effective tool to identify the nitroproteins and effective biomarkers in human astrocytomas, clarifying the biological roles of oxidative/nitrative stress in the pathophysiology of astrocytomas, functional characteristics of nitroproteins in astrocytomas, nitration-mediated signal pathway network, and early diagnosis and treatment of astrocytomas. The results finds that these nitroproteins are enriched in mitotic cell components, which are related to transcription regulation, signal transduction, controlling subcellular organelle events, cell perception, maintaining cell homeostasis, and immune activity. Eleven statistically significant signal pathways are identified in astrocytoma, including remodeling of epithelial adherens junctions, germ cell-sertoli cell junction signaling, 14-3-3-mediated signaling, phagosome maturation, gap junction signaling, axonal guidance signaling, assembly of RNA polymerase III complex, and TREM1 signaling. Furthermore, protein tyrosine nitration is closely associated with the therapeutic effects of protein drugs, and molecular mechanism and drug targets of cancer. It provides valuable data for studying the protein nitration biomarkers, molecular mechanisms, and therapeutic targets of astrocytoma towards PPPM (3P medicine) practice.


Clinical characteristics and epidemiology of gliomas
Glioma originates mainly from neurointerstitial cells.The nervous system tumors account for 2% of total tumor incidence, of which glioma incidence accounts for 40-50% of intracranial tumors and 90% of malignant tumors of the central nervous system.Astrocytoma is the most common type of gliomas, and astrocytes also occupy an important position in the central nervous system, participating in the control of extracellular environmental homeostasis, synaptic activity, and neuronal development [1,2].Reactive astrocytes can protect against brain injury, but severe, prolonged, and rapid responses can lead to a massive inflammatory response, causing massive neuronal death [3].The 2016 World Health Organization (WHO) classification of central nervous system tumors integrated molecular and histological features for the first time to promote a more accurate classification of these tumors [4], the fifth edition of the WHO Central Nervous System (CNS) in 2021 classifies gliomas into pediatric and adult types, which are adult-type diffuse glioma, diffuse low-grade glioma, pediatric-type diffuse highgrade glioma, confined astrocytic glioma, glial neuronal and neuronal tumors, and ventricular canal tumors, respectively.According to the histological morphological features, gliomas can be divided into astrocytoma, oligodendroglioma, ventricular meningioma, and mixed glioblastoma (GBM).Astrocytoma accounts for 20-40% of gliomas.According to the grade of tumor and its growth site, pathological pattern and biological behavior, astrocytoma is divided into four grades from 1 to 4. Grades 1 and 2 gliomas are less malignant and mostly benign, such as hairy cell astrocytoma.Grade 4 gliomas are more malignant and are more common in GBM, with rapid proliferation, increased vascularity, and infiltration into the surrounding brain tissue [5].Traditional treatments for gliomas include the safest surgical resection, assisted by a combination of radiotherapy, chemotherapy, and immunotherapy.

Importance of protein tyrosine nitration in gliomas
Tyrosine nitration exists in many diseases including cancer, inflammation, and neurodegeneration.There is significant protein nitration in human glioma, especially astrocytoma (grade 1), oligodendroglioma (grade 2), and highly malignant GBM (grade 4) [6].Nitric oxide (NO) is the source of oxidative/nitrosative stress, and also the nitrogen source for the formation of nitrating agent.It is synthesized by L-arginine through nitric oxide synthases (NOS), and the activity of NOS can be induced by relevant glioma cell lines such as astrocytes [7].Three subtypes of NOS, including neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), can all produce NO, and eNOS and nNOS are expressed in nerve cells and endothelial cells of glioma, respectively [8][9][10].iNOS can induce a large number of NO production in a variety of cells, especially astrocytes under pathological conditions.NO generates reactive nitrogen species (RNS) and reactive oxygen species (ROS) such as peroxynitrite (ONOO − ) and nitrogen dioxide (NO 2 ) in the presence of oxidants such as superoxide radicals, transition metal centers, and hydrogen peroxide (H 2 O 2 ), inducing the nitration of amino acids, proteins, lipids, and nucleic acids [11][12][13].ONOO − and heme peroxidase pathways are the main pathways for the formation of tyrosine nitration in protein.ONOO − is the main mediator of tyrosine nitration, introducing a nitro group into the carbon atom at the tyrosine residue to form 3-nitrotyrosine (3-NT) [14].NO 2 − and H 2 O 2 form 3-NT under the catalysis of heme peroxidases horseradish peroxidase or lactoperoxidase (LPO).Stable 3-NT is detected by a variety of techniques, and is used as a relatively specific marker of oxidative stress in humans.Protein tyrosine nitration changes the amino acid redox potential, pKa, hydrophobic, and other key properties, so tyrosine nitration can change the protein structure and conformation, the catalytic activity of the enzyme, and the sensitivity of protein hydrolysis, thus changes the biological function of protein (Fig. 1) [15,16].For example, nitrate peroxide-mediated tyrosine nitration results in the loss of functional activities such as manganese superoxide dismutase (MnSOD), succinyl-CoA: 3-oxoacid CoA-transfer, protein kinase C, neurofilament-L, cytochrome P450 2B 11, actin, prostacyclin synthase, and tyrosine hydroxylase [17][18][19][20][21][22][23].Tyrosine nitration also increases the activity of weak or absent functions previously present in the nitroprotein, such as ONOO − activation of the microsomal glutathione S-transferase 1 enzyme [24].The conformation of cytochrome c was changed by its nitration, which in turn caused an increase in its peroxidation activity.In addition, nitrated cytochrome c was also able to resist the reduction of antichymic acid, damaging respiratory function, nitration of cytochrome c can also inhibit apoptosis, and fibrinogen nitration accelerate fibrin clot formation [25][26][27].Tyrosine nitration may also affect the immune activity of the tumor.For example, autoprotein containing nitrotyrosine is recognized as an immunogenic autoantigen, causing a very strong anti-autoimmune response [28], signal transducer and activator of transcription 1 (STAT1) is highly expressed in normal brain tissue, while it is poorly expressed, or not expressed in human glioma.STAT1 may inhibit the proliferation of glioma cells, promote apoptosis, and inhibit tumor angiogenesis.Studies have shown that nitration of STAT1 inhibits the immune response of dendritic cell [29].RNS in the tumor microenvironment induces nitration of C-C motif ligand 2 and results in loss of its ability to attract tumorspecific cytotoxic T lymphocyte [30].Tyrosine nitration provides a new pathway for immunotherapy.

Relationship of tyrosine nitration and tyrosine phosphorylation
There is an interaction between post-translational modifications (PTMs).It is taken for example, nitrosation, phosphorylation, and S-nitrosation.S-nitrosation is the transfer of nitration equivalents from the donor to thiol as a functional intermediate, and the number of cysteine nitrosation proteins is at least 10 times that of tyrosine nitrosation proteins [16].It is similar to other modifications such as acetylation, ubiquitination, and phosphorylation in some aspects, and can regulate the function of a variety of proteins.After the cysteine residue at the active site of the enzyme is S-nitrosed, the enzyme activity is inhibited [31].Protein tyrosine nitration inhibits tyrosine phosphorylation; for example, nitration of tyrosine residues within B56δ inhibits modification of B cell lymphoma-2 (Bcl-2) phosphorylation [32].Tyrosine nitration reduced the strength of tyrosine-phosphorylated protein in endothelial cells, but the nitrated bovine serum albumin (BSA) was degraded by the lysates, indicating that tyrosine nitration not only interfered with phosphorylation but targeted protein for degradation [33].In addition, tyrosine phosphorylation participated in all kinds of signaling pathways, such as oxidative stress, cell growth, proliferation, and migration.For example, Src family kinase phosphorylation in glioma Dock180 tyrosine residue Y722 mediates the activation of epidermal growth factor receptor VIII and Ras-related C3 botulinum toxin substrate 1 signals, and promotes the migration and survival of GBM cells [34].On the contrary, tyrosine phosphorylation can also block the formation of tyrosine free radicals, further blocking tyrosine nitration [35].However, studies have also shown that the nitration of Tyr might not prevent the phosphorylation of Tyr because the consensus sequence of Tyr phosphorylation is different from that required for nitration in vivo [36].

The effects of tyrosine nitration on structure and functions of a protein
Nitration of protein is selective, and there are many factors that affect the nitration selectivity of tyrosine residues.For example, (i) physical and chemical properties of the environment: The amount of nitration products produced by ONOO − depends on pH, and the nitration reaches the maximum under the physiological condition of pH 7.5, with the pKa values of 6.8 and 7.9-8.01,respectively [37]; the reaction rate of NO with O 2 was significantly accelerated in a hydrophobic environment, and biofilm and other hydrophobic sites are important sites for the formation of NO and NOderived reactive species [38]; carbon dioxide/bicarbonate not only enhances the nitration of the aromatic ring system, but also resists the inhibition of nitration by antioxidants, and partially inhibits the oxidation of mercaptans.These functions could accelerate the nitration of tyrosine.Transition metal ions such as Cu 2+ , Fe 3+ , Fe 2+ , and myeloperoxidase accelerate ONOO − -mediated nitration [39].(ii) Close to that generates part of the nitrating agent: tyrosine nitration occurs in a specific functional domain in a nitroprotein of a specific cell type, ONOO − and NO 2 are highly unstable, and the half-life is short, so tyrosine nitration is more likely to occur near the generating part of the nitrating agent; for example, mitochondria are the key part for generate nitrite,

3-nitrotyrosine
Fig. 1 The formation of nitrotyrosine and its effect on protein functions and more nitroproteins are available [40].MnSOD was more susceptible to inactivation by ONOO − -mediated Tyr34 nitration than by NO 2 -dependent tyrosine [15].(iii) protein structure: at least 95% of the tyrosine residues in protein are buried.Souza JM et al. developed a model to discuss the factors affecting nitration, which consisted of three proteins with similar size but different tyrosine contents and different three-dimensional structures.The results showed that the position of tyrosine on the ring structure and the exposure of aromatic ring to the molecular surface were the factors determining tyrosine nitration, the tyrosine level of free 3-NT is up to 30 time higher than that of protein, thus demonstrates that that hydrophobicity of the tertiary structure of protein protects certain amino acids from nitration [41].Two nitration residues, Tyr44 and Tyr129, were detected by Luo Y et al. on p16 protein induced by nitrite peroxide, and Tyr44 was more easily nitrated [42].The results of chemical environment analysis of the two residues indicated that steric hindrance might be the structural determinant of tyrosine nitration sequence [42].(iv) Intra-molecular electron transfer and electrostatic force close to tyrosine residue: Intramolecular electron transfer between cysteine residue and tyrosine radical hinders tyrosine nitration but induces protein nitrosation.Studies have shown that amino acids near tyrosine residues closer to negatively charged amino acids, such as glutathione, will make protein tyrosine nitration is enhanced, and near the positively charged amino acids, such as cysteine residues, tyrosine radicals and Cys intramolecular electron transfer between the residues to reduce protein nitration, also studies have shown that Cys residues are more easily oxidized, so Tyr nitration is reduced [43].However, there is a different understanding of nitration near methionine residues, with studies suggesting that methionine residues stimulate oxidation and nitration of tyrosine residues [44].Methionine was also considered to be a readily oxidizable amino acid that reduced the nitration of Tyr [45,46].Neighboring glutamic acids cause ONOO − attack on specific tyrosine [47].Protein nitration is a dynamic and reversible process.The ONOO − -mediated protein nitration occurs mainly in the mitochondria, which can regulate the electron transport chain.The 3-nitrostyryl can be completely eliminated within 20 min of hypoxia, and then reduced to nitration within 5 min after reoxygenation.This protein tyrosine nitration/denitration process regulates the cell signal transduction process, which is linked to oxidative stress.In a short time, it can play a role in antioxidant defense and regulation of mitochondrial metabolic rate.Long-term hypoxia and reoxygenation will lead to excessive nitration, which may cause circulatory shock, myocardial ischemia, and other injuries [48,49].Denitration enzyme can modify the activity of protein containing nitrotyrosine residue and facilitate signal transduction, histone is the most important nitrating protein in cancer, histione 1.2 can be used as the substrate of denitrating enzyme to purify and characterize the activity of denitrating enzyme, the activity of denitrating enzyme can reduce the immunoreactivity of nitrotyrosine [50].

Nitroproteomics as an effective tool to identify nitroprotein biomarkers
This paper reviews the present situation of nitroproteomics of human astrocytoma, including the biological role of oxidative/nitrative stress in the pathophysiology of astrocytoma, key characterization of nitropeptide in human astrocytoma by mass spectrometry (MS), identification of nitroprotein in human astrocytoma by MS/MS, enrichment of endogenous nitropeptide/nitroprotein, cancer treatment based on nitration, according to the previous research results in the laboratory, some functional characteristics of nitroprotein in astrocytoma and the signal pathway network of nitration in astrocytoma were studied, and the prospect of this research was put forward.Protein tyrosine nitration is involved in the entire pathophysiological process of astrocytoma.Clarification of protein tyrosine nitration will benefit discovery of molecular biomarkers for insights into accurate molecular mechanism and identification of therapeutic targets for targeted therapy, and construction of molecular models for patient stratification, prediction, diagnosis, and prognostic assessment of astrocytoma in the framework of predictive, preventive, and personalized medicine (PPPM; 3P medicine).

Working hypothesis in the framework of PPPM
We hypothesize that nitroproteome and nitration-mediated signaling pathway networks are significantly different in astrocytoma tissues relative to control brain tissues.The differentially nitrated proteins, nitration sites, and nitrationmediated signaling pathway networks, coupled with other omics data and clinical data, offer great promise for insight into the accurate molecular mechanisms of astrocytoma, discovery of effective therapeutic targets and drugs, and determination of reliable nitration-related biomarkers for patient stratification, predictive diagnosis, prognostic assessment, and personalized medical services of astroglioma in the framework of PPPM.

Biological role of oxidative/nitrative stress in astrocytoma pathophysiology
One of the characteristics of cancer cells is a high level of oxidative stress [51].Oxidative stress is generated by RON/ ROS, which is involved in the biochemical process of cancer cell oxidative/nitration stress.Low level of RON/ROS promotes the proliferation, survival, and anti-stress ability of cancer cells by activating signal transduction pathways, such as nuclear factor-kappa B (NF-κB) pathway induced by RON/ROS [52].H 2 O 2 mediates cell proliferation, differentiation, and migration [53].High levels of RON/ROS promote cancer cell death [54].Studies have shown that nuclear factor E2-related factor 2 (Nrf2) over-activation in GBM can be used as the prognosis of glioma, Nrf2 antioxidant function can reduce ROS production, and NRF2 deficiency in cancer cells inhibits nitration caused by oxidative stress to the detriment of cancer progression [55][56][57].In addition, ROS induces DNA damage, leading to tumor mutation; ROS can also activate mitochondrial kinetics-related enzymes, such as photostudylinositol-3-kinase and mitogen-activated protein kinase, and regulate biological energy to support tumor proliferation, invasion, and migration [58].
ROS can also induce iNOS, which induces NO production.Many signaling pathways affect iNOS and NO, and thus affect nitration.For example, the p38 mitogen-activated protein kinases (MAPK) pathway regulates the production of iNOS and the activation of nicotinamide adenine dinucleotide phosphate oxidase in astrocytes through oxidative stress to generate ONOO − , and its specific inhibitor can attenuate ONOO − production [59].Typical intermediate filaments of astrocytes exist in many nitrotyrosine-labeled glial processes, and the NO donor induces tyrosine nitration in astrocytes and neurons [60].NO can regulate a variety of pathophysiological process of tumors, including angiogenesis and dilation of gliomas, increased vascular permeability, immunosuppression, free-radical damage adjacent to normal tissue, and therapeutic resistance [61]; for example, NO promotes the proliferation of glioma cells [62].Inducing the expression of the vascular endothelial growth factor gene in GBM induces angiogenesis [63].Broholm H et al. found that in gliomas, especially astrocytomas, nNOS promotes the progression of malignant tumors more than eNOS and iNOS, and may be a useful indicator for glioma grading.After eNOS increases the production of NO in glial tumor endothelial cells, it can cause cerebral edema [10,64].NO activates c-Jun N-terminal kinase and its downstream pathway, and induces the increase of Bcl-2 antagonist/killer (Bak) and Bcl-2 interacting mediator levels, which in turn affects mitochondrial permeability.Bak can also mediate the downstream pathway of oxidative stress, leading to apoptosis of GBM cells [65,66].In GBM, NO induces the nitration of Tyr-152 and Tyr-66 residues in p65 subunits of NF-κB to inactivate the biological activity of NF-κB, which thereby achieves anti-inflammatory effects and inhibits the survival, proliferation, invasiveness, and angiogenesis of cancer cells [67].NO can also affect the immune activity in glioma.The results of Oda T et al. showed that high level of NO in glioma inhibited the production of IL-8, and excessive NO regulated the production of chemokines to reduce tumor inflammatory reaction [68].Interferon gamma increases the activity of iNOS in astrocytomas, and the resulting NO enhances the expression of MHCII antigen, further affecting the immune response [69].
ONOO − is formed by the reaction of O 2 • with •NO.It is the main strong oxidant for ROS to participate in the nitration, and can directly oxidize the transition metal center, mercaptan and other biomolecules with strong electrons, or rapidly react with CO 2 to form secondary free radicals to indirectly participate in the oxidation reaction.O 2 • reacts with SOD to form competition with the formation of ONOO − .ONOO − generated under oxidative/nitrative stress can nitrate wild-type p53 protein of glioma, resulting in loss of binding ability of p53 DNA, loss of p53 transcriptional activity, imbalance of downstream p21 expression, degradation of p53 protein, and inactivation of tumor inhibition pathway, thereby promoting tumor growth [70,71].In human neuroblastoma, peroxidase inhibits the phosphoinositide signaling pathway and affects the activation of transcription factors, thus affecting the death of cancer cells, and it transiently affects the content of several protein phosphotyrosine, in which tyrosine phosphorylation of the p120 Src substrate is significantly increased, whereas astrocytoma cells are resistant to the action of ONOO − [72].ONOO − is also involved in the apoptotic process of glioma cells.For example, capsaicin can induce the apoptosis of glioma cells.However, glioma cells exposed to capsaicin are found to have more nitrotyrosine proteins.The ONOO − has been proven to be the reason that mediates this phenomenon [73].In addition, ONOO − also promotes the oxidation and nitration of guanine in DNA, and the nitrated guanine nucleotide, 8-nitrscGMP, in glioma cells can be used as an electrophilic second messenger to participate in the regulation of ROS signal transduction, and can also induce Nrf2 activation and antioxidant enzymes to protect cells from cytotoxic effects [74,75].
3-NT is considered to be an ideal biomarker for oxidative stress in vivo due to its stability.When the cells cannot tolerate the metabolic pathway of D-glucose, it will lead to oxidative stress.The 3-nitro-L-tyrosine in neuroblastoma cells exposed to D-glucose is increased, and the tubulin is downregulated.3-nitro-L-tyrosine doped into microtubule protein destroyed the microtubule network [76].

Mass spectrometry characterization of nitropeptides
Proteomics studies the synthetic protein of the translation process, a new technique for identification and analysis of all protein in biological samples.Proteomics method does not only give the overall level of analysis of the nitration protein of cells or tissues but can also avoid the exogenous nitrating agent or no nitration, so the method is used to study the mechanism of tyrosine nitration selectivity [77].MS is a leading tool for the study of proteomics with the characteristics of high sensitivity, resolution, and accuracy.It characterizes PTM based on the specific molar mass change of amino acid covalent modification products.The MS method can be used to modify the oxidation/nitration in peptide mixtures or in amino acid hydrolysates.However, only MS analysis of modified peptides can detect specific amino acid residues [78].In recent years, MS has been successfully applied to directly identify the modification of tyrosine residues in protein mediated by active nitrogen species.Electrospray ionization (ESI)-MS/MS and matrix-assisted laser desorption/ ionization time-of-flight MS (MALDI-TOF) are the most commonly used MS techniques for studying tyrosine nitration of peptides and protein [39].
MALDI-MS and ESI-MS can analyze and characterize 3-NT-containing peptides [79].MALDI-TOF-MS directly measures molecular weight and can be used to analyze relatively simple peptide mixtures.ESI-MS high-resolution mass spectrometry analyzes multi-charge signals generated by electrospray, and then deconvolutes the signals to obtain accurate molecular weight values, which can be used to analyze complex samples [80].Notably, ultraviolet (UV)-MALDI weakens or eliminates peptide modifications when analyzing nitropeptides [39,81,82].Petersson AS et al. used MALDI-and ESI-MS to analyze the mass spectra of nitrated bovine serum albumin and angiotensin II peptide, respectively.The results showed that the signal abundance of nitropeptide detected by MALDI-MS was low, and ESI-MS could identify half of the nitropeptide of bovine serum albumin, indicating that neither of the two detection methods was a reliable method for detecting the nitration of protein.
The combination of precursor ion scanning and mass spectrometry was a sensitive and specific detection method for nitropeptide [83].Collision-induced dissociation (CID) is a common dissociation method for ESI-MS/MS, this method results in poor PTM characterization of the sequencing sequence [81].However, electron-capture dissociation (ECD) and electron-transfer dissociation (ETD) will not crack the unstable side chain of PTMs, and become the first choice to characterize PTMs, but the double-charged ECD containing 3-NT peptide is difficult to generate sequence fragments, the ECD of tri-charged 3-NT-containing peptides only produced some single-site charge sequence fragments.The ECD of nitropeptides had the characteristics of multiple loss of water, •OH, and ammonia, which also showed that ECD was not the preferred method to characterize nitration [84].Mikhailov VA et al. analyzed the nitrated lysozyme, cytochrome C, and myoglobin with the top-down method.The results showed that the number of CID and infrared multiple photon dissociation cleaved near the nitration site was larger than that of ECD.ECD provided more complete nitration fragments, and ECD could study complete nitration proteins [85].MS-based metastable atom-activated dissociation (MAD) method: The MAD-MS method was used to detect synthetic peptide GPLEnYGFAKGPLAK, nitroangiotensin II (DRVnYIHPF), generate a-,b-,c-,x-,y-, and z-types ions, so that a complete peptide sequence is obtained and accurate positioning of the nitration sites is realized; and the MAD can also distinguish I/L residues in the nitropeptide and induce free-radical ion chemistry to determine nitration peptides [86].
Fiore G et al. identified human glioma α-tubulin protein trypsin nitropeptide by MALDI-MS method, the coverage of α-tubulin primary structure sequence was 46%, and two signals with mass difference of 45 KDa were detected at m/z 1763.9 and 1718.9, peptides 216-229 were detected at m/z 1763.9, peptide mixtures analyzed by LC/MS/MS with sequence coverage of 53%, a total of 10 Tyr residues were identified, including Tyr272, Tyr83, Tyr262, Tyr108, Tyr103, Tyr312, Tyr319, Tyr357, Tyr393, and Tyr408, and the α-tubulin specific nitration site Tyr224 in human glioma was identified for the first time by peptide mass fingerprinting (PMF) analysis, although some tyrosine residues remained undetectable [6].Peng F et al. used LC-ESI-qTOF and LTQ-Orbitrap Velos MS/MS analysis method to identify the purified peptide mixture derived from human astrocytoma.A variety of nitropeptides including nitropeptides ITFDDnYIAC*C*VK from sorcin (Fig. 2) and nitropeptide GHnYTEGAELVDSVLDVVR derived from tubulin beta-2A chain, tubulin beta-2B chain, tubulin beta-3 chain, and tubulin beta chain were identified [87].
The characterization of nitropeptides is the fundamental step to deeply investigate endogenous nitroproteins in human astrocytoma.

Enrichment of endogenous nitropetides/ nitroproteins in human astrocytoma
Although MS is the key technology for characterizing nitropeptide/nitroprotein and determining nitrotyrosine sites, the level of intracellular endogenous nitropeptide/nitroprotein is usually very low, effective enrichment method is required for large-scale identification of nitropeptide/nitroprotein before MS analysis [88].Peng F et al. preferentially enriched nitroproteins by two-dimensional gel electrophoresis (2DGE), followed by protein blotting of nitrotyrosine [87].The proteins containing 3-NT could be enriched by immunoprecipitation of nitroproteins with anti-nitrotyrosine antibodies, which were then purified and analyzed by ESI-MS/MS to identify multiple proteins [89]; however, using specific antibodies for immunoprecipitation in advance has the disadvantage of time-consuming, expensive, and low efficiency.In order to solve this problem, a calculation method for finding nitration sites has been developed, such as GPSYNO 2 [90] and iNitro-Tyr [91] are used for tyrosine nitration; however, these algorithms predict potential modification sites inaccurately and cannot fully characterize the biological characteristics of these sites, Xie Y et al. developed DeepNitro using the method of depth algorithm, a new type of software to predict nitration [92].On that other hand, other enrichment methods are developed, such as a biotin-labeled method is developed, 3-NT is reduced into 3-aminotyrosine (3-AT) by using a strong reduce agent, a cleavable sulfo-NHS-SSbiotin label is connected on the aminotyrosine, protein is digested by trypsin, then purified on an avidin column, and finally identified by MS, and peptides containing 3-NT and protein can be selectively enriched [93,94].An alternative method for enriching nitrotyrosine-derived mercaptopeptides with thiopropyl sepharose beads is highly efficient, as the method identifies the non-enriched in vitro nitration of human histone H1.2, BSA, the contrast percentages of the nitrotyrosine-derived peptides identified with LC-MS/ MS and mouse brain homogenate samples were only 9%, 9%, and 5.9%, while those of the enriched samples were 91%, 62%, and 35%, and 150 kinds of nitropeptides were identified from mouse brain homogenate with an error rate of only 3.3%; however, the above two methods can only identify exogenous single nitroprotein but cannot identify endogenous nitroproteins [95].In order to identify endogenous nitrates, Abello N et al. blocked all amines before reduction of 3-NT, followed by aminotyrosine biotinylation, strong cation exchanger removal of free biotin, and protein column enrichment of labeled peptides.This improved method promoted the identification of endogenous nitropeptides.This method successfully enriched tyrosine-nitrated angiotensin II in BSA trypsin peptide matrix [96].Through a method which converts nitro groups into high-level uorinted moities and then selectively enriches nitropeptides/ nitroproteins based on fluorine-fluorine interaction, 28 kinds of nitroproteins are detected from liver cancer cells, which provides a new way for further research on tyrosine nitration of protein [97].Dremina   sulfonyl)pyrrolidine-3,4-diol, labeled the residues in the 3-NT fluorescence-derived peptide and performed borate affinity enrichment.The method was tested successfully in C 2 C 12 cells.The enrichment method provided the possibility of 3-NT peptide sequence-specific MS analysis and had potential application value in protein nitration characterization [98].

Identification of nitroprotein in human astrocytoma by tandem mass spectrometry
3-NT can be detected with antibody-based quantitative methods such as enzyme-linked immunosorbent assay (ELISA) assay, immunohistochemistry, high-performance liquid chromatography (HPLC), electrochemical detection, and mass spectrometry.Detection of 3-NT levels in serum and synovial fluid of patients with rheumatoid arthritis in 1994 by HPLC supported NO-mediated injury, and high-throughput and high-resolution protein components were identified by LC-MS/MS [99].In the same year, mouse monoclonal antibodies and rabbit polyclonal antibodies were developed to recognize nitroprotein, which was subsequently detected in human coronary atherosclerotic lesions [100,101].Then, a competitive ELISA was used to quantify 3-nitro-L-tyrosine in human nitroproteins and nitropeptides, such as nitrated serum albumin and nitrated fibrinogen [102].Immunocytochemical localization of nitrotyrosine and constitutive in the brain of protein-imprinted rats demonstrated significant nitrotyrosine labeling in astrocytic processes [60].Liquid chromatographic electrochemical detection is a highly sensitive method to measure 3-NT, which offers the possibility of estimating protein nitration [103], the sensitivity of HPLC and electrochemistry for the detection of 3-NT in protein is 100-fold greater than that using UV/VIS [104].2DGE-MS is a commonly used technique to identify protein containing 3-NT.The combination of 2DGE and western blot allows high-throughput recognition of specific targets for protein nitration, and the combination of MS allows identification of nitroproteins [105].LC-MS, gas chromatography (GC)-MS/MS, LC-MS/MS, and GC-MS based on MS method, especially GC-MS/MS and LC-MS/MS, are considered as the gold standard for defining the reference value of endogenous substances.LC/MS mainly analyzes polar and high molecular weight compounds such as peptides and protein [106].Lee et al. used LC/ESI-MS/MS method to detect the mass shifts of +45 Da produced by the nitration of angiotensin II and tetranitro-methane in vitro, and characterized the nitration [107].According to MS/MS data, the range of free 3-NT is 0.5-3 nm in healthy normal people and the range of protein-related 3-NT is 0.4-1.6 × 1: 10 6 [108].Robinson RA et al. used a combination of precursor isotope labeling and combined precursor isotopic labeling and isobaric tagging to analyze the 3-NT modified protein by blocking the N-terminal of tyrosine residues and trypsin peptide with "light" and "heavy" labeled acetyl groups, then reducing the 3-NT to 3-AT, and finally derivatizing the labeled 3-AT peptide with either iTRAQ or TMT complex reagents, which was the first method to identify nitropeptides using quantitative protein omics to obtain accurate quantitative information [109].The α-tubulin was identified by LC-MS/MS method in the 55-KDa nitroprotein band of astrocytoma [6].In human astrocytoma tissue, Peng F et al. detected 57 nitroprotein positive spots by nitrotyrosine western blot and 18 nitroproteins were identified by MS/MS analysis in astrocytoma tissues, including sorcin, tubulin beta chain, probable G-protein coupled receptor 52, Ras-related protein Rab-8B, coiled-coil domain-containing protein 105, Ig kappa chain V-I region WAT, and regulating synaptic membrane exocytosis protein 1 [6] (Table 1), and the result of nitrated sorcin was confirmed with immunoprecipitation and western blot (Fig. 3).Multiple tumor suppressor gene (p16) inhibits cell proliferation, and the positive rate of p16 is gradually reduced in grade 1-4 astrocytomas, showing strong tumor inhibition.Two mononitration residues Tyr129 and Tyr44 in p16 were identified by HPLC-MS/MS [42].These nitroproteins identified in astrocytoma provide the protein tyrosine nitration atlas of astrocytoma.

Functional characteristics of nitroproteins in astrocytoma
The -NO 2 group introduced into tyrosine phenol ring is an electron-withdrawing group, which will reduce the electron density of phenol ring, and the reduced electron density will weaken the binding force between receptor and its ligand or enzyme and its substrate, thus affecting the protein function.Studies have shown that nitroproteins are associated with some organelles, such as the outer mitochondria membrane, nuclear membrane, microtubules, the nucleolus, and tubovesicles and synaptic vesicles [60].The above conclusions were similar to the 18 nitroproteins identified by Peng F et al. [87] in human astrocytoma tissue (Table 1), and each protein had a certain function; for example, nitrosorcin is associated with multidrug resistance and metastasis of astrocytomas, and tubulin, Tau, and tubulin cofactor B (TCoB) are the first three proteins of the discovered nitrocytoskeleton.Fiore G et al. characterized the endogenous nitroproteins in astrocytomas with immunohistochemistry and proteomics, and found that tubulin was the target of nitration in the tumors, and α-tubulin was nitrated on Tyr224, which was the drug-binding domain in α-tubulin [6].The incorporation of 3-NT into tubulin causes morphological changes, decreased viability, growth inhibition, and nuclear involvement in glioma cell lines [110].EBI is  the main modulator of microtubules (MT), and the detyriasis/tyrosination cycle can be performed in GBM to inhibit endothelial cell migration [111], nitro-β-tubulin blocks cell morphological differentiation and participates in cytoskeleton and cell migration [87,112].The study by Katsetos CD et al. revealed aberrant expression and complex formation of III β-tubulin and γ-tubulin in astrocytomas, suggesting that this aberrant expression and interaction of tubulin is related to the degree of tumor deterioration in gliomas and might be a promising tumor marker [113].Strong iNOS was present in tau-positive astrocytes.Tau regulated cytoskeletal proteins by nitration and serine/threonine phosphorylation, and TCoB was nitrated on Tyr-64 and Tyr-98.After nitration, TCoB not only weakened microtubule synthesis, but also antagonized TCoB phosphorylation at residues Ser-65 and Ser-128 [114,115].Ras superfamily regulates cell proliferation and differentiation, which is related to protein transport and cytoskeleton construction.Studies have shown that the activation of RAS is common in malignant astrocytomas [116].Activation of Ras can also activate MAPK and other downstream pathways, resulting in excessive cell proliferation and microvessel formation.Ras-related protein on chromosome 22 can inhibit the growth, proliferation, and migration of astrocytoma cells, and induce their atypical apoptosis [117].Peroxidation regulates PTM of amino acid residues of presynaptic and postsynaptic proteins in synaptosomes, and tyrosine phosphorylation in prominent bodies is increased by 50-500 μM ONOO − and decreased by more than 500 μM [118].Modification of specific tyrosine residues regulates the association of synaptic vesicles proteins and affects the extracellular endocytosis cycle [119].In addition, Sacksteder CA et al. identified 29 types of nitroproteins from mouse brain, including related proteins in mitochondria, energy metabolism, inflammation, cytoskeleton, and dopamine synthesis [120].Souza J M et al. found that tyrosine hydroxylase in mouse brain was nitrated, which caused the loss of enzyme activity and decreased dopamine level in mouse brain [41].
Nitroprotein may affect apoptosis [121].Alpha-synuclein is a soluble protein expressed presynaptic and perinuclear in the CNS.Over-expression of alpha-synuclein in human neuroblastoma cells increases ROS production, increases mitochondrial protein nitration, decreases mitochondrial transmembrane potential, and promotes apoptosis [122].
Tyrosine-induced proteasome degradation of nitroprotein leads to the loss of specific biological functions [123]; for example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat astrocytes is a sensitive nitroprotein, and low-dose ONOO − can nitrate GAPDH, which increases the sensitivity of proteasome degradation [124].

Nitration-medicated signaling pathway networks in astrocytomas
Tyrosine nitration in protein is selective, only a few is nitrated, and only a few tyrosine residues in each protein are modified [125].Protein tyrosine nitration alters astrocyte function in a manner that is dependent on N-methyl-D-aspartatic acid receptor, Ca 2+ and NOS [126].The activation of Ca 2+ /calmodulin/MAP kinase kinase/ERK pathway was induced to inhibit the proliferation of astrocytoma cells [127].Interleukin1b (IL-1b) is a cytokine of astrocyte proliferation, IL-1b mediates NO/Ca 2+ /CaM/ ERK signaling pathway in astrocytoma cells [128,129].It can be seen that protein tyrosine nitration is involved in the signal pathway of astrocytoma.In this study, 18 nitroproteins previously identified by laboratory researchers in human astrocytoma by gel electrophoresis and tandem mass spectrometry were searched by UniProt database (https:// www.unipr ot.org) (Table 1), and then these nitroproteins were subjected to gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) and disease enrichment analysis by ClusterProfiler (https:// yulab-smu.top/ biome dical-knowl edge-mining-book/ refer ences.html# ref-wu_ clust erpro filer_ 2021), further analysis of signal pathway, upstream regulator, diseases & functions and molecular network of nitroproteins in astrocytoma by Ingenuity Canonical Pathways (IPA).Analysis of significantly enriched GO terms found that these nitroproteins are enriched in 4 kinds of cell components (CCs), including intercellular bridge, mitotic spindle, spindle and microtubule (Fig. 4A, Supplementary table 1); and in 17 molecular functions (MFs), including structural constituent of cytoskeleton, GTPase activity, GTP binding, guanyl nucleotide binding, guanyl ribonucleotide binding, transmembrane transporter binding, GTPase regulator activity, nucleoside-triphosphatase regulator activity, RNA polymerase III general transcription initiation factor activity, siRNA binding, G protein-coupled photoreceptor activity, GTPase activating protein binding, interleukin-1 receptor binding, photoreceptor activity, MHC class I protein binding, GTPase activator activity, and pattern recognition receptor activity (PRR) (Fig. 4A, Supplementary table 1).KEGG pathway analysis of those nitroproteins identified multiple signaling pathways, including Salmonella infection, gap junction, phagosome, motor proteins, pathogenic Escherichia infection, Parkinson disease, Prion disease, Huntington disease, Amyotrophic lateral sclerosis, Alzheimer disease, and pathways of neurodegeneration-multiple diseases (Fig. 4B).Salmonella infection and Gap junction are the most relevant enrichment pathways of nitroproteins in human astrocytoma.Finally, we analyzed network plot and hierarchical clustering of enriched diseases of 14 nitroproteins in astrocytoma (Fig. 4C, D).The results of IPA enrichment showed that it was mainly enriched in remodeling of epithelial adherens junctions, germ cell-sertoli cell junction signaling, sertoli cell-sertoli cell junction signaling, 14-3-3-mediated signaling, phagosome maturation, Gap junction signaling, breast cancer regulation by stathmin1, axonal guidance signaling, assembly of RNA polymerase III complex and TREM1 signaling (Supplementary Figure 1, Supplementary Table 2).According to different diseases and functions, these nitroproteins (Supplementary Table 3) and molecules in their enriched networks can be divided into three branches: (1) cellular movement, developmental disorder, neurological disease; (2) cell morphology, cellular development, and hair and skin development and function, (3) connective tissue disorders, developmental disorder, and hereditary disorder (Table 2; Fig. 5).Through IPA, we also found some upstream regulators targeting these nitrated proteins (Supplementary Table 4).
CC results show that nitroproteins are enriched in mitotic cell components, such as intercellular bridge to promote material transport between two divided daughter cells [130].Studies have shown that PTMs regulates mitotic spindle to promote mitosis [131].Spindle localization is involved in cell and organ differentiation, etc. Integrin is an immunerelated adhesion molecules, and cell adhesion mediated by it is very important for the formation of spindle [132].Microtubule participates in cell movement, intracellular material transport, can form spindle and participate in cell division.MF results showed that nitrifying protein in astrocytoma was related to transcription regulation, signal transduction, controlling subcellular organelle events, cell perception, maintaining cell homeostasis, and immune activity.Interleukin-1 receptor mediates immune signal transduction, MHC class I protein presents immunogen peptide, and PRR activity participates in innate immune regulation signal transduction, which shows that nitroprotein may regulate signal transduction through immune pathway.Nitrated tubulin β class I gene (TUBB) identified at nY106 site in our laboratory participated in all signal pathways enriched by KEGG.TUBB is proved to be highly expressed in many cancers, and mediates cell cycle, proliferation, migration, invasion and metastasis, which plays an important role in cancer progress [133].For example, TUBB can mediate the survival of neuroblastoma, which is closely related to the poor prognosis of liver cancer and kidney cancer, and may also become a biomarker for breast cancer prediction.TUBB is resistant to many chemotherapeutic drugs such as vincristine and paclitaxel [133].According to the clinical data of GBM patients, some studies screened the differentially expressed genes related to survival, and made bioinformatics analysis.It was found that TUBB was one of the core genes that affected the chemical sensitivity of GBM to Semustine [134].Nitroproteins of human astrocytoma identified in our laboratory is also involved in other complex signal pathways, gap junction is involved in the development of cancer in different ways.There are gap junctions in astrocytes, which regulate the growth of cancer cells by controlling gene expression depending on gap-junction communication or independent ability, establishing communication through endothelial barrier can enhance the migration, adhesion, and diffusion of cancer cells to matrix, and gap junction is very complicated in various functions [135,136].Autophagy degrades some worn organelles and redundant or defective protein on a large scale, fuses with lysosomes, and works in cell homeostasis, autophagy can also alleviate cell stress caused by hypoxia and metabolic disorder, inhibit tumor progression in cancer cells, and promote the survival of cancer cells under adverse conditions [137].
IPA-enriched pathways participate in autophagy and immune-related pathways, such as coronavirus inducing autophagy accumulation, preventing autophagy-lysosome fusion and enhancing virus replication [138].The process of phagocyte maturation is an important innate defense mechanism for macrophages to resist pathogen infection.It is generally believed that autophagy system starts toll-like receptor (TLR) signaling of phagocytes to promote phagocyte maturation, and ROS is necessary for autophagy protein recruitment to phagocytes [139].TREM1 signaling and TLR signaling cooperate to produce pro-inflammatory factors and promote phagocytosis [140].RNA polymerase III participates in the synthesis of RNA in homologous recombination and repair of double-stranded breaks, and RNA polymerase III senses DNA virus, which is transcribed to induce the generation of type I interferon and participate in innate immune response [141].IPA-enriched pathways interact with oxidative stress, such as coronavirus enhancing redox stress, inducing inflammation and apoptosis, damaging cells and tissues and affecting the central nervous system.Many redox mechanisms can regulate coronavirus replication, such as phagocytosis and apoptosis [142].iNOS induced shortterm interruption of adherens junctions, and NO/NOS regulated adherens junction kinetics of sertoli-germ cells through cGMP/PRKG pathway [143].NOS regulates the kinetics of tight junction in sertoli cells through NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway [144].ROS regulates axonal-directed signal transduction to cope with nervous system injury [145].IPA-enriched pathway affects the development of astrocytoma, and 14-3-3 regulates the phosphorylation of various kinases, affects the progress of cell cycle, binds to various proteins, and participates in vesicle transport, signal transduction and DNA replication.The combination therapy of some drugs can induce cell cycle arrest and apoptosis through AKT/14-3-3 pathway to inhibit the progress of breast cancer [146].In astrocytoma, 14-3-3 is highly expressed.Some studies have shown that the mechanism of astrocytoma escaping from apoptosis may be the upregulation of 14-3-3 isomer expression, and it is considered that 14-3-3 protein may be the target of future research on astrocytoma treatment [147].Overexpression of 14-3-3β can inhibit the phosphorylation of β-catenin, induce its nuclear translocation, and further enhance the transcription of oncogene, thus regulating the proliferation of astrocytoma [148].Stathmin1 is a microtubule regulatory protein, which participates in the assembly and disassembly of mitotic spindle and is related to cancer metastasis.The high expression of Stathmin1 can be used as a biomarker of breast cancer.Studies have shown that Stathmin1 is also highly expressed in malignant astrocytoma, which promotes the invasion of GBM cells [149,150].
Previous studies have shown that the nitration sites of nine nitroproteins identified from human pituitary adenoma are all located in the functional domain where nitration occurs [151].In this study, protein domains and motifs of 17 nitroproteins (Table 1) were searched by Motifscan https:// myhits.sib.swiss/ cgi-bin/ motif_ scan, and 15 proteins were found, some nitration sites are located in the domain of nitrated protein, phosphorylation site exists upstream or downstream of all nitration sites.We found the nitration site nY110 of H7C0C0 was located at the tyrosine kinase phosphorylation site KSGERH1Y (Supplementary Figure 2).NetPhos-3.1 (https:// www.expasy.org/ search/ unipr ot) identifies the amino acid position where phosphorylation occurs in the peptide chain and on which amino acid it occurs, and tyrosine phosphorylation can occur at all nitration sites except P80362; for some nitroproteins, nitration and phosphorylation might compete the same tyrosine residue to affect protein functions, including the nitrated sorcin, tubulin beta chain, tubulin beta-3 chain, tubulin beta-2A chain, tubulin beta-2B chain, coiled-coil domain containing protein 105, isoform 2 of signal induced proliferation associated 1-like protein 2, isoform 2 of Arf-GAP with SH3 domain, ANK repeat and PH domain containing protein 2, isoform 2 of grainy head like protein 1 homolog, and isoform 2 of Toll-like receptor 9 (Supplementary Figure 3).

Cancer therapy based on protein nitration
Nitration of protein tyrosine is a free radical-mediated reaction, which can reduce protein tyrosine nitration in cancer by reducing the level of ONOO − ; for example, given antioxidants, the potential antioxidant effect of hydrogen sulfide (H 2 S) can significantly inhibit ONOO − -mediated tyrosine nitration and protein oxidation and nitration in human neuroblastoma.As a new neuromodulator, H 2 S deserves further study [152].As an adapter molecule, p130 cas is phosphorylated at the adhesive spot, and participates in the process of cell migration, survival, invasion, etc.The p130 cas has been identified as a new target protein for nitration induced by ONOO − donor SIN-1 in human neuroblastoma, and tyrosine nitration may destroy p130 cas tyrosine phosphorylation, thus affecting the development of glioma [153].NO can be used as a tumor killer, which not only produces resistance in the chemotherapy process of astrocytoma cell therapy, but also in radiotherapy.Its mechanism is to irradiate p53 protein, induce NO synthesis, and start the intracellular signal transduction system.Understanding the molecular dynamics of NO is helpful to establish a new therapeutic viewpoint [154].The enzyme nNOS enhances the damage caused by temozolomide chemotherapy in astrocytoma and GBM, further study of nNOS is helpful to reveal its potential role in the anti-tumor treatment of astrocytoma [155].Enzymes iNOS and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) mediate the production of RNS/ROS in the brain, and inhibitors of the SFK-iNOS-NOX2 signaling axis are potential targets for neurological diseases [156].Several studies have considered the pathophysiological effects of chronic systemic NOS inhibition on the growth and blood flow of glioma.It is considered that chronic systemic NOS is mainly anti-tumor in glioma model, which provides a new method for the treatment of glioma and is worthy of further study [157].
Avastin® (bevacizumab) is a monoclonal antibody, which can inhibit vascular endothelial growth factor, and is widely used to treat cancer.Food and Drug Administration (FAD) has approved it for recurrent GBM.Bevacizumab is regarded as the most potential therapeutic antibody, but it has serious side effects.Some studies have identified various tyrosine nitration sites in avastin by LC-ESI-MS/MS and HCT and LTQ Orbitrap MS based on 2DGE [158].There is no exception, adriamycin (ADR) is an effective broadspectrum anti-tumor antibiotic, which is clinically used for the treatment of various malignant tumors.However, ADR also leads to nitration of protein [159].To sum up, nitration of drugs is a serious challenge in pharmaceutical industry.In addition, some drugs can induce or inhibit the mRNA expression of astrocytoma mediated by interferon-gamma, LPS, tumor necrosis factor-alpha and interleukin 1 beta [160,161].Drugs can also affect the nitration of tumor.For example, morphine can inhibit tyrosine nitration induced by ONOO − in human GBM [162].Dasatinib (Sprycel), as a selective inhibitor of protein tyrosine kinase, plays an immunomodulatory role [163].Angiotensin receptor blockers, renin-and angiotensin-converting enzyme-inhibitors and statins can reduce vascular oxidative stress [164].Some drugs have become very promising cancer prevention drugs by promoting tyrosine nitration (Table 3).
Synthetic molecules also play a role in therapy.Hydroxyphenyl nitrones of microwave-assisted synthesis is a new molecule synthesized by Chavarría C et al.The combination of this molecule with the antioxidant fragment can stably generate free radicals with nitrone, showing antioxidant activity, inhibiting tyrosine nitration of alpha-synuclein protein mediated by ONOO − , improving cell activity of injured human neuroblastoma, and preventing human neuroblastoma from tyrosine nitration, which has potential therapeutic effect [171].Nagaraj S et al. found that tyrosine nitration of T-cell receptor (TCR)/CD8 complex destroyed the binding of CD8 + T cells and pMHC dimer, thereby affecting the interaction between TCR/CD8 and pMHC and its conformational activity.Tyrosine nitration can change TCR contact residues and affect T cell recognition, or change the MHC contact position, thus affecting T cell recognition.Tyrosine nitration provides a new tumor T cell tolerance mechanism and a new idea for tumor immunotherapy [172,173].
Atukeren P et al. evaluated apoptotic and adhesion molecules, nitric oxide metabolites, cytokines, myeloperoxidase, levels of lipid peroxidation, SOD, and myeloperoxidase in plasma and tissues of GBM multiforme and normal controls.Only SOD dismutase was significantly declined in tumors, and intercellular adhesion molecule-1 was the highest expression in astrocytes.And the higher the expression quantity of vascular cell adhesion molecule-1 and plasma intercellular adhesion molecule-1, the higher the level of nitrite/nitrate, plasma 3-NT, plasma interleukin-1beta also related to plasma nitrite/nitrate.It shows that there is no balance between apoptosis and anti-apoptosis mechanism, which affects tumor progress, and further reveals that targeted single molecule has little effect in cancer treatment [174].

Future perspectives
In the appearance of 2DGE technology made it possible to study the total protein of an organism on a whole level.In 1994, Wilkins and Williams first proposed the concept of "proteome".Then the new discipline of proteomics came into being.In 2001, the Human Proteome Organization (HUPO) was established and the Human Proteome Project (HPP) was launched.The completion of sequencing of the human genome project in 2003 provided people with materials and tools to study human diseases.Subsequently, more and more genome and proteome information were used to serve prediction, prevention and personalized medical treatment of diseases, which laid the foundation for the development of the 3P medical era.Although great progress has been made in proteomics, the MS analysis method for studying the nitropeptide/nitroprotein of human astrocytoma is still very complicated.Nitration is common in glioma and cancer [6].It is of great significance to study the molecular mechanism of nitration in glioma and to prevent, predict, and personalize the treatment of glioma by studying the nitroprotein of human astrocytoma more extensively and deeply.In recent years, the research on nitroprotein has provided an important target for the development of 3P medicine in the twenty-first century, and it is expected to become an important marker for detecting diseases and provide a new direction for treating diseases.For example, studies have shown that myoibrillar isoform of creative kinase (MM-CK) is a highly sensitive target for the formation of 3-NT, and it is believed that protein nitrification is involved in the energy disorder of myocardial cells and the energy shuttle mechanism of CK in vivo [175].Amyloid β (Aβ) is nitrated at tyrosine 10 (3NTyr10-Aβ), and the 3NTyr10-Aβ induces plaque formation.NOS 2 (iNOS) inhibitors can significantly reduce Aβ deposition, 3NTyr10-Aβ is considered as a potential target for iNOS treatment [176].In terms of prevention and prediction, some studies have shown that exogenous spermidine can effectively block DNA nitration and prevent renal ischemia-reperfusion injury [177].
Studies have used immunohistochemical method to detect the levels of iNOS and nitrotyrosine in oral squamous cell carcinoma (OSCC), and found that iNOS mRNA, iNOS protein and nitrotyrosine all increased, which were related to staging, metastasis, survival, and recurrence of OSCC.It is considered that nitrotyrosine is an important index to identify the prognosis of OSCC [178].
We propose the following aspects to expand and strengthen the studies on nitroproteomics in astrocytoma: (i) Develop new methodology for nitroproteomics of astrocytoma.The untargeted high-throughput quantitative proteomics strategy is applied to the study of human astrocytoma nitroproteins, such as isobaric tag for relative absolute quantification/tandem mass tags (iTRAQ/TMT), label-free strategies, data independent acquisition (DIA), protein parallel reaction monitoring (PRM), protein/peptide quantification, etc. iTRAQ/TMT can quantify the proteome of multiple samples by using various isotope tags to label the peptide after proteolysis and combining with high-precision MS analysis.However, label-free strategies can realize protein quantification without isotope labeling, which preserves the authenticity of the sample to the greatest extent.The peptide fragments of protein were analyzed by LC-MS.The above two methods have been widely used in number of fields such as, biological disease markers, molecular mechanism of biological disease occurrence and development, drug action target, drug action mechanism and so on [179].The basic process of DIA is different from that of iTRAQ/TMT and label-free.After the protein sample is hydrolyzed, it has to undergo polypeptide desalting, low-temperature drying and re-dissolution, and finally a pool sample is generated.The sample and the actual sample are collected by GPF-DIA and single-run respectively, and the data is analyzed by using Prosit model and encyclope-DIA platform.This method can collect all ions and fragment maps with high quantitative accuracy, and can simultaneously detect all target proteins.PRM preferentially selects the parent ion of the target peptide, then fragmenting the parent ion, and finally detecting all the fragment information in the window of the selected parent ion with orbitrap analyzer, which can be used to verify the modified quantitative proteomics and realize the relative and absolute quantitative analysis of the target protein/peptide, with high specificity, accuracy, reliability, sensitivity, and flux.(ii) Build a 3D structure of a nitroprotein.The threedimensional structure of protein is very important for the nitration of tyrosine residues [41].If the 3D structure of human astrocytoma nitroprotein can be constructed, its biological function can be explained intuitively, and small molecule drugs suitable for the 3D structure can be designed.Because the nitration abundance of protein tyrosine is very low, effective enrichment methods are very important.
(iii) Strengthen the application of enrichment method.At present, the enrichment of human astrocytoma nitroprotein is limited to the protein blot of nitrotyrosine based on 2DGE, and many enrichment methods introduced in this paper have not been applied [88,179].(iv) Strengthen individualized medical care.Due to the complexity of the process and detection methods of nitropeptide/nitroprotein, individualized medical treatment may provide a new method for studying patients with astrocytoma.(v) According to the nitration site of differentially nitrated protein in astrocytoma, a cell line or mouse model with stable mutation site was constructed, and its upstream influence mechanism and downstream function changes were further detected to find possible therapeutic targets for astrocytoma.(vi) The plasma of patients with different grades of astrocytomas was studied with nitroproteomics, and different nitroproteins, nitration sites, and nitrationmediated signaling pathway networks were searched, so as to deeply understand the accurate molecular mechanism of astrocytoma, find effective therapeutic targets and drugs, and find reliable biomarkers.
Because obtaining plasma requires no major trauma, the price is low, and the most important thing is to reduce potential complications and realize individualization [180].The specific molecular patterns in the body fluid such as plasma, and tear fluid are systemic effects for disease prevention and staging [180].(vii) The relationship of protein tyrosine nitration and tumor inflammation.Tumor inflammation is important pathophysiological characteristics, and is involved in protein tyrosine nitration.Protein nitration biomarkers derived from tumor inflammation might offer the systemic effects for cancer prevention of astrocytoma [181].(viii) The relationship of protein tyrosine nitration and angiogenesis.Tumor angiogenesis is another important pathophysiological characteristics in astrocytoma, which is associated with eNOS and Nol, and even protein tyrosine nitration.Protein nitration biomarker derived from vascular component has the systemic effects in advanced cancer management of astrocytoma [182].Moreover, protein nitration is also involved in mitochondrial health component which is systemic effects in cancer prediction and prevention [183].

Conclusions and expert recommendation in the context of 3P medicine
This article reviews the present situation and prospect of nitroproteomics of human glioma, mainly astrocytoma, GO/KEGG analysis showed that the molecular function of nitroprotein in human astrocytoma was mainly enriched in structural constituent of cytoskeleton, and GTPase activity; cell components are mainly enriched in chromaffin granule, and trans-Golgi network transport vesicle; biological processes are mainly enriched in microtubule-based process, and microtubule cytoskeleton organization; and signal pathways are mainly concentrated in Salmonella infection, and gap junction.The signal pathway network of nitration in astrocytoma was preliminarily explored.It provides a basis for studying the biological significance of tyrosine nitration in astrocytoma and its correlation with the pathogenesis of glioma.
We recommend strengthening the studies on nitroproteomics in astrocytoma.It can identify protein tyrosine nitration profile, astrocytoma-specific nitroproteins and nitration sites, and nitration-mediated signaling pathway network changes, and discover protein nitration biomarkers.These data will benefit indepth insight into molecular mechanism and discovery effective therapeutic targets for astrocytoma, and also establishment of nitration biomarkers for patient stratification, predictive diagnosis, prognostic assessment, and personalized medical services for astrocytoma in the framework of 3P medicine (Fig. 6).
This nitroptoeomics in combination with other proteomics, transcriptomics, and clinical data in astrocytoma offers the PPPM innovation in the following three aspects.
(i) Predictive approach.Tyrosine nitration is an important oxidative stress-related protein modification in human body, and its abnormal changes exist in entire pathophysiological process of astrocytoma, crossing occurrence and development of astrocytoma.Nitroproteomics data in combination with other proteomics data, transcriptomics data, and clinical data can establish nitration-related predictive diagnosis model and prognostic assessment model to innovate the predictive approach of astrocytoma.(ii) Targeted prevention.Nitration-mediated signaling pathway network alteration and their regulation benefit to deeply understand molecular mechanism of astrocytoma, and discover effective therapeutic targets and drugs, which directly contribute to the targeted prevention and therapy of astrocytoma.(iii) Personalization of medical services.Integrative analysis of nitroproteomics, other proteomics, transcriptomics, and clinical data can construct nitrationrelated molecular biomarkers for patient stratification of astrocytoma, and personalized predictive diagnosis, prognostic assessment, and treatment of astrocytoma to realize personalized medical services.Fig. 6 The schematically current approach of nitroproteomics in overall astrocytoma management versus advanced one in the context of 3P medicine In summary, nitroproteomics can identify nitroproteins, nitration sites, and nitration-mediated signaling pathway changes, which is an innovative area in the research field of astrocytoma.Moreover, integrative analysis of nitroproteomics, other proteomics, transcriptomics, and clinical data can reveal nitration-based survival, hub molecules, and drugs against the key nitroproteins in astrocytoma towards predictive diagnosis, targeted prevention, and personalized medical service.This present article demonstrates an innovative, nitration-based state of the art contributing to the paradigm shift from reactive medicine to PPPM in human astrocytoma.

Fig. 3
Fig. 3 Expression and nitration levels of sorcin in gliomas relative controls.A The protein expression level of sorcin in astrocytomas (Stages I, II, III, and IV) relative to controls (N), identified with western blot.B Tyrosine nitration level of sorcin in stage IV of astro-

Fig. 5
Fig. 5 Nitration-mediated molecular networks in astrocytoma, identified with Ingenuity Pathway Analysis (IPA).A Network 1 is cellular movement, developmental disorder, and neurological disease.B Network 2 is cell morphology, cellular development, and hair and skin

Table 2
Nitration-mediated molecular networks in astrocytoma, identified with ingenuity pathway analysis (IPA)

Table 3
Drugs and tyrosine nitration