**3. Progress in proteomics**

It is recognized that gene expression changes does not reflect the changes in protein expression within the cell. Better tools are needed to accurately probe the protein activities and levels. Protein arrays are arguably underachieving their potentials as they are perceived not as precise as DNA arrays. Functional proteomics still holds great promises that it could result in greater understandings of the mechanism of disease. The goals proteomics of diseases are to improve molecular classification of diseases and to discover molecular biomarkers for their diagnosis, treatment target and following up [66–68]. Better tools are needed to accurately calculate the protein activity levels. Protein arrays (used synonymously with protein microarrays or protein biochips) are one of the solutions to the high throughput study of protein interaction networks or immune reactivity [69–71]. Mass spectroscopy (MS) [72], relies mainly

**27**

*Proteomics Research and Its Possibility of Application in Endometriosis*

on the mass-to-charge ratios to distinguish different proteins. It is widely used as a discovery tool of diagnostic, prognostic, and therapeutic protein biomarkers. Moreover, it does not need to identify molecules. And protein arrays depend on the availability of specific recognition molecules. MS-based methods can simultaneously screen numerous proteins, but its sensitivity is approximately 1 ng/mL. Emerging protein arrays based on magneto-nanosensors enable to study many proteins to proteins interactions simultaneous easily with sensitivity low than about 1 pg/mL. Proteomic researches mainly includes the 2-DE reference map and a database by proteomic comparison between healthy persons and patients. Comparing to western blot, 2-DE can be used

to find out autoantigens that may induced autoantibodies in some diseases.

limited because only the molecules represented on the array can be identified.

In the past few decades, the paradigm of biomarker research has shifted from a hypothesis-driven approach to a discovery-driven approach. Mass spectrometry and separation techniques and proteomics methods have been fully developed and have been common. Particularly when two-dimensional liquid chromatography/ tandem mass spectrometry, or two-dimensional gel electrophoresis (2-DE) and matrix laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) combination, followed by database search (sequence tags or peptide mass fingerprints) are widely used. These methods greatly increase the comprehensive-

Identify disease-associated antigens can elicit through immune responses by combining protein separation (2-DE, gel-free separation), immunological detection (Western blotting) and MS, or combining immunocapture and MS [78]. Proteins derived from cells or tissues (e.g., cancer cells) are separated by 2-DE, and antigen proteins are detected by applying patient serum, which may include disease-specific antibodies followed by enzyme-labeled secondary antibodies. In order to identify

Protein arrays with antibodies are developing a tool for rapid measurement of abundance of proteins expression. It makes the possibility to screen the aimed proteins changes in different diseases and biological processes. In most cases, this approach depends on exposing serum samples from patients to an ordered array of antigens, capturing those antibodies that bind the antigens on the arrays. Immobilization of proteins on the surface of arrays and neutralizing reactive areas after the immobilization are important practical issues in protein array. Many different types of proteins arrays such as antibody arrays and peptide arrays have been reported [73]. It starts research in breast cancer and leukemia [74, 75]. Recently, one of the protein array technologies is the magneto-nanosensor array where giant magnetoresistive (GMR) [76] sensors are used to quantitatively measure analyte of interest proteins which are labeled with magnetic nanoparticles (MNP). Another emerging protein array technology is Nucleic Acid Programmable Protein Arrays (NAPPA) [77], which have thousands of protein features directly expressed by nucleic acids on array surface. More attentions have been paid to the role of protein arrays in medicine. They can be used for early detection of diseases, diagnosis of stages, stratification of patients, and prediction of therapeutic effects, and are increasing realization of the vision of personalized medicine. For cytokine measurements, protein arrays should be improved in both functional sensitivity and probe density. Till now, the arrays have two major drawbacks: first, they are biased, because antigen selection is based on their potential to play a role in disease. Secondly, the analytical comprehensiveness of this technique is

*DOI: http://dx.doi.org/10.5772/intechopen.81850*

**3.1 Protein arrays**

**3.2 Mass spectrometry (MS)**

ness of protein identification.

*Proteomics Research and Its Possibility of Application in Endometriosis DOI: http://dx.doi.org/10.5772/intechopen.81850*

on the mass-to-charge ratios to distinguish different proteins. It is widely used as a discovery tool of diagnostic, prognostic, and therapeutic protein biomarkers. Moreover, it does not need to identify molecules. And protein arrays depend on the availability of specific recognition molecules. MS-based methods can simultaneously screen numerous proteins, but its sensitivity is approximately 1 ng/mL. Emerging protein arrays based on magneto-nanosensors enable to study many proteins to proteins interactions simultaneous easily with sensitivity low than about 1 pg/mL. Proteomic researches mainly includes the 2-DE reference map and a database by proteomic comparison between healthy persons and patients. Comparing to western blot, 2-DE can be used to find out autoantigens that may induced autoantibodies in some diseases.

#### **3.1 Protein arrays**

*Molecular Bases of Endometriosis - The Integration Between Research and Clinical Practice*

**2.5 Proteins involving in peritoneal fluid and plasma of endometriosis**

cytokines and chemokines, later amplifying their effects.

cells recognition involved in endometriosis pathogenesis.

between pro- and anti-apoptotic factors [65].

**3. Progress in proteomics**

Most of researches have demonstrated that macrophages, lymphocytes, endometrial cells and mesothelial cells are able to produce cytokines and inflammatory mediators such as ILs [3, 46, 47], TNF-a [48], PGF2, PGE2 and thromboxane B2 [49], MCP-1 [50], RANTES [51], eotaxin [52], GROa [47], SDF1 [53], and MIF [50, 54]. The main process is leading to the stimulation of endometriotic cells proliferation and adhesion to ectopic sites, angiogenesis, and stimulation of the release of other

Macrophages can amplify the activity of COX-2 and PGE2, which results in VEGF stimulation in endothelial cells from endometriotic lesions, together with factor StAR, association with an increased estrogen level in the endometrial tissue [55–57]. PGE2 suppresses the activity of phagocytes, that allowing endometriotic implants formatting [58]. Estrogens and PGE2 can induce FGF-9 expression which can further activate endometrial cells proliferation, paralleling to the stimulation of angiogenesis and apoptosis inhibition at the same time. Lymphocytes are involved in various cytokines production with potential role in endometriosis lesions implanting. Whether in vitro study or in human endometriosis, Th2 cells of peritoneal fluid were shown to stimulate the secretion of IL-4 and IL-10. NK cell-mediated cytotoxicity, commonly manifested by lymphocytes adherence to endometrial cells through LFA-1 — ICAM-1 pathway and their presentation as targets to NK cells, may fail in endometriosis [59, 60]. This indicated the possible mechanism that sICAM-1 may bind to LFA-1 expressing in lymphocytes can prevent endometrial

TNF-a is a typical pro-inflammatory cytokine produced by macrophages, which can exhibit high levels in serum and peritoneal fluid of patients with endometriosis [61, 62]. Recent studies have shown that TNF-a-induced activation of IKKb complex leads to the initiation and progression of endometriosis by enhancing the survival rate of ectopic epithelial cells rather than stromal cells and not eutopic epithelial cells [63]. MIF, another cytokine, has been shown a high level in the peritoneal fluid, in serum samples, and in peritoneal macrophages, its secretion being regulated by estrogens in endometriosis [85]. MIF can stimulate endothelial cell proliferation, endometriotic lesions survival, expressing VEGF, IL-8, PGE2, COX-2, MCP-1, aromatase, and resulting back in stimulating TNF-a in endometrial cells [18, 64]. That is why in experimental models, MIF antagonist significantly reduces lesions size of endometriosis by inhibiting cell adhesion, tissue remodeling, angiogenesis, and inflammation, in addition to routine alteration of the balance

It is recognized that gene expression changes does not reflect the changes in protein

expression within the cell. Better tools are needed to accurately probe the protein activities and levels. Protein arrays are arguably underachieving their potentials as they are perceived not as precise as DNA arrays. Functional proteomics still holds great promises that it could result in greater understandings of the mechanism of disease. The goals proteomics of diseases are to improve molecular classification of diseases and to discover molecular biomarkers for their diagnosis, treatment target and following up [66–68]. Better tools are needed to accurately calculate the protein activity levels. Protein arrays (used synonymously with protein microarrays or protein biochips) are one of the solutions to the high throughput study of protein interaction networks or immune reactivity [69–71]. Mass spectroscopy (MS) [72], relies mainly

**26**

Protein arrays with antibodies are developing a tool for rapid measurement of abundance of proteins expression. It makes the possibility to screen the aimed proteins changes in different diseases and biological processes. In most cases, this approach depends on exposing serum samples from patients to an ordered array of antigens, capturing those antibodies that bind the antigens on the arrays. Immobilization of proteins on the surface of arrays and neutralizing reactive areas after the immobilization are important practical issues in protein array. Many different types of proteins arrays such as antibody arrays and peptide arrays have been reported [73]. It starts research in breast cancer and leukemia [74, 75]. Recently, one of the protein array technologies is the magneto-nanosensor array where giant magnetoresistive (GMR) [76] sensors are used to quantitatively measure analyte of interest proteins which are labeled with magnetic nanoparticles (MNP). Another emerging protein array technology is Nucleic Acid Programmable Protein Arrays (NAPPA) [77], which have thousands of protein features directly expressed by nucleic acids on array surface.

More attentions have been paid to the role of protein arrays in medicine. They can be used for early detection of diseases, diagnosis of stages, stratification of patients, and prediction of therapeutic effects, and are increasing realization of the vision of personalized medicine. For cytokine measurements, protein arrays should be improved in both functional sensitivity and probe density. Till now, the arrays have two major drawbacks: first, they are biased, because antigen selection is based on their potential to play a role in disease. Secondly, the analytical comprehensiveness of this technique is limited because only the molecules represented on the array can be identified.

#### **3.2 Mass spectrometry (MS)**

In the past few decades, the paradigm of biomarker research has shifted from a hypothesis-driven approach to a discovery-driven approach. Mass spectrometry and separation techniques and proteomics methods have been fully developed and have been common. Particularly when two-dimensional liquid chromatography/ tandem mass spectrometry, or two-dimensional gel electrophoresis (2-DE) and matrix laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) combination, followed by database search (sequence tags or peptide mass fingerprints) are widely used. These methods greatly increase the comprehensiveness of protein identification.

Identify disease-associated antigens can elicit through immune responses by combining protein separation (2-DE, gel-free separation), immunological detection (Western blotting) and MS, or combining immunocapture and MS [78]. Proteins derived from cells or tissues (e.g., cancer cells) are separated by 2-DE, and antigen proteins are detected by applying patient serum, which may include disease-specific antibodies followed by enzyme-labeled secondary antibodies. In order to identify

immunogenic proteins, the corresponding spots are separated from the gel and gels were digested. MS or tandem mass spectrometry is used for analysis and then analyzed by peptide fingerprinting or sequence tags method.

### *3.2.1 Isotope-coded affinity tagging multidimensional LC-MS*

LC–MS makes identification and quantification of target protein possible in a large and complex sample and most of the time it may not be achieved in the clinical work. However, it will be useful when proteins are limited in a sample and at present this method will be considered as a supplement to 2-DE gel [79, 80].

### *3.2.2 MALDI-MS*

Antigen analysis of immunocapture MS is derived from the immobilization of antibodies in the serum of patients. Almost all antibodies are captured on protein A or G, which is a bacterial derived protein with specific affinity for the Fc domain of the antibody. Protein mixtures (cell or tissue lysates) are applied to a column or bead immobilized with antibodies to capture specific antigens of antibodies present in patient samples. Finally, the proteins were identified by MALDI-TOF-MS or surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS). In SELDI-MS, the protein is enzymatically cleaved into a peptide mixture and undergoes MS. It can be a fully automated system allowing high-throughput and rapid identification. MS afford a method to identify a protein even from a complex mixture of proteins [81, 82]. It is preferred and an applicable for a pure protein or a single spot for 2-DE gel.

### *3.2.3 Surface-enhanced laser desorption/ionization (SELDI-MS)*

Chemical defined or antibodies-coated protein biochip arrays for rapid protein detection. This system is used when small amount of samples is available. Very high surface expression of the immunoglobulin binding protein (proACTR) as the antigen capture and transfer reagent [83]. ProACTR can immobilize the antibody through the Fc region of antibody, and allows for higher capture capacity than antibody-coated beads. Captured antigens can be directly transferred to a platform for MS analysis (SELDI-TOF-MS). It starts its diagnosis research with post-translationally modified proteins and high-throughput technique in breast cancer, lung cancer and prostate cancer. Unfortunately, it does not allow reliable protein sometimes [84, 85]. To the best of our knowledge, immunoproteomics with proACTR has not yet been applied to profile antigens associated with a certain disease, but mainly to the quantification of a single target [86, 87].
