**5.1. Idiopathic pulmonary fibrosis**

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disorder characterized by continuous production of extracellular matrix that alters parenchymal lung structure and effective gas exchange. Cigarette smoking, exposure to agriculture and farming, livestock, wood and metal dust, stone, and silica have been associated with significantly increased risk of IPF. The etiopathogenesis of the disease is not completely understood, and the prognosis is very poor. In light of this, proteomic studies aimed at the discovery of molecules involved in triggering and progression of this disorder could help to a better understanding of its physiopathology. As expected, all proteomic approaches to study this disorder which involved the use of an electrokinetic procedure have been performed on BALf.

Kim et al. [28] compared by 2-DE and nano-LC-MS/MS the proteomes of BALf from patients affected by IPF and healthy subjects. An increase of haptoglobin and a decrease of α1-antitrypsin, α1-antichymotrypsin, macrophage capping protein, angiotensinogen, hemoglobin chain B, apolipoprotein (Apo A-I), clusterin, protein disulfide isomerase A3, immunoglobulin, and complement C4A were observed in IPF subjects. That a local treatment with Apo A-I could be effective against the development of experimental lung fibrosis was shown in mice. The main goal of the study performed by Ishikawa and colleagues [29] was to discover new markers for both IPF and chronic obstructive pulmonary disease (COPD). By comparing the 1-DE and 2-DE profiles of BALf and IS of IPF and COPD patients with those of controls, they showed a reduction in the levels of hemoglobin α (Hbα) and hemoglobin β (Hbβ) monomers and complexes only in IPF patients. Furthermore, MS analyses revealed that a modification at Cys 105 (most likely the site involved in complex formation) of Hbα was responsible for the lack of the Hbα complexes formation in IPF patients. 2-DE, MS, Western blotting, and ELISA were applied by Ohlmeier et al. [30] to lung tissues and BALf of IPF, COPD, and alpha-1 antitrypsin-deficient (AATD) patients with the aim to investigate the different receptors for advanced glycation end product (RAGE) isoforms. Their analyses revealed a decrease of the full-length RAGE (FL-RAGE) and its C-terminal processed variant (cRAGE) in the lung tissues of IPF and COPD patients but not in AATD. The endogenous secretory RAGE (esRAGE) level was reduced in IPF but remained unchanged in COPD. The 2-DE proteomic study by Hara et al. [31] aimed at searching for BALf biomarkers allowing to distinguish IPF from other fibrotic interstitial pneumonias. Their study showed that S100A9 protein (or calgranulin B, a member of the calcium-binding S100 protein family) was upregulated in IPF patients compared to patients with other fibrotic interstitial diseases and healthy controls. Based on these findings, S100A9 was thought to be a good candidate biomarker to discriminate IPF from other fibrotic interstitial pneumonias. The research group by Landi et al. [32] used the electrokinetic approach to compare the proteome of BALf from patients affected by four interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis, pulmonary Langerhans cell histiocytosis, fibrosis associated to systemic sclerosis) with that of controls (smokers and nonsmokers). The proteins they identified (i.e., plastin 2, annexin A3, 14-3-3ε, and S10A6) could be directly or indirectly related to the pathophysiology of the different interstitial lung diseases. The same research group compared the profile of BALf proteins from IPF patients, never-smokers, and smokers to understand which protein(s) could be potentially related to disease progression and pathogenesis. Their findings showed that angiotensin system maturation, renin angiotensin-aldosterone system, heme metabolism, coagulation system, response to hypoxia, oxidative stress, and iron transport were the metabolic pathways involved in disorder [33]. The same group also investigated the molecular patterns and their variability in familial and sporadic IPF patients through a differential proteomic analysis based on 2-DE and MS [34]. It was observed that, while in familial IPF, the upregulated proteins were those involved in wounding and immune responses, coagulation system, and ion homeostasis, and those involved in the oxidative stress response were upregulated in sporadic IPF.

Silva et al. [35] have used a 2D-based proteomic approach to analyze BALf of patients affected by sarcoidosis and chronic beryllium disease (CBD) with the aim of comparing the protein profiles at the site of active inflammation. Briefly, the differentially expressed proteins were highlighted by 2D-DIGE and identified by MALDI-TOF. In accordance with the ongoing chronic inflammation response in lungs of these patients, most of these proteins were

The Role of One- and Two-Dimensional Electrophoretic Techniques in Proteomics of the Lung

http://dx.doi.org/10.5772/intechopen.75042

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While being a well-known risk factor for the development of various interstitial lung diseases, smoking is generally not related to the occurrence of sarcoidosis. To evaluate better the influence of cigarette smoking on the proteome of BALf from sarcoidosis patients and to study the pathogenetic mechanism of the disease, Landi et al. [36] investigated by 2-DE the protein profile and network of BALf from sarcoidosis patients, smokers (SC), and nonsmokers (NSC) used as controls. MALDI-TOF MS revealed that 34 spots contained unique proteins involved in lipid, mineral, and vitamin D metabolism and immune regulation of macrophage function. These findings confirmed that, differently from other ILDs, the expression profile of proteins

Systemic sclerosis (SSc) is a disease of unknown origin characterized by increased deposition of collagen and other extracellular matrix proteins in skin and multiple internal organs. About 70% of SSc patients develop a severe and progressive lung fibrosis, ILD being the main cause of mortality. Nevertheless, the mechanisms involved in the onset and progression of fibrosis remains unknown. To clarify the causative role of ILD in SSc, Shirahama et al. [37] analyzed the protein profiles of BALf from patients affected by SSc with and without pulmonary fibrosis (SSc-fib+ and SSc-fib−). 2-DE combined with MALDI-TOF MS was the method of choice. The authors showed that, among other proteins identified, α2-macroglobulin, α1-antitrypsin, and pulmonary surfactant protein A were upregulated in SSc-fib+ patients, while α2 heat shock protein (HSP) and glutathione S-transferase (GST) were downregulated in the same patients compared to SSc-fib ones. These results suggested that these proteins could be potentially

Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting 0.5–1.0% of the adult population worldwide. Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) occurs in 10–30% of RA patients and is associated with increased mortality in up to 10% of RA patients. The pathogenetic mechanisms of RA-ILD are still unknown. The most common pulmonary patterns in RA-ILD are usual interstitial pneumonia (UIP) and nonspecific interstitial pneumonia (NSIP). Nevertheless, the less common organizing pneumonia (OP) and lymphocytic interstitial pneumonia are not as rare as hypothesized. The proteomic profiles of UIP and OP were compared by Suhara et al. [38] by analyzing BALf of patients with 2-DE and nano-LC-ESI-MS/MS. The levels of the fragmented proteins gelsolin and Ig kappa chain C region were found to be significantly higher in UIP patients compared to those with an OP pattern. Conversely, the levels of α1-antitrypsin, C-reactive protein (CRP), haptoglobin β, and surfactant protein A (isoform 5) were significantly higher in the OP than in the UIP patterns.

from sarcoidosis patients was more comparable to that of NSC than of SC.

**5.3. Lung fibrosis associated with systemic sclerosis**

involved in the development of ILD in SSc patients.

**5.4. Rheumatoid arthritis-associated lung disease**

immune-related proteins.

#### **5.2. Sarcoidosis**

Sarcoidosis is a multisystem disorder characterized by the formation of epithelioid cell granulomas. Even though lungs, lymph nodes, and eyes are most commonly involved, any organ or system of the body can be affected. Patients with genetic susceptibility undergoing persistent exposure to unknown inhaled antigens may develop an excessive immune response mediated by antigen-presenting cells (APC) that can trigger the pathological mechanisms of the disease. However, its etiology still remains an enigma and a challenge for researchers and clinicians due to its unknown, variegated, and unpredictable presentation. The recent state of the art relative to the application of proteomic studies aimed at gaining insights into the mechanisms of the disease is reported in this section.

Silva et al. [35] have used a 2D-based proteomic approach to analyze BALf of patients affected by sarcoidosis and chronic beryllium disease (CBD) with the aim of comparing the protein profiles at the site of active inflammation. Briefly, the differentially expressed proteins were highlighted by 2D-DIGE and identified by MALDI-TOF. In accordance with the ongoing chronic inflammation response in lungs of these patients, most of these proteins were immune-related proteins.

While being a well-known risk factor for the development of various interstitial lung diseases, smoking is generally not related to the occurrence of sarcoidosis. To evaluate better the influence of cigarette smoking on the proteome of BALf from sarcoidosis patients and to study the pathogenetic mechanism of the disease, Landi et al. [36] investigated by 2-DE the protein profile and network of BALf from sarcoidosis patients, smokers (SC), and nonsmokers (NSC) used as controls. MALDI-TOF MS revealed that 34 spots contained unique proteins involved in lipid, mineral, and vitamin D metabolism and immune regulation of macrophage function. These findings confirmed that, differently from other ILDs, the expression profile of proteins from sarcoidosis patients was more comparable to that of NSC than of SC.
