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

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

28 Electrophoresis - Life Sciences Practical Applications

the oxidative stress response were upregulated in sporadic IPF.

mechanisms of the disease is reported in this section.

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

**5.2. Sarcoidosis**

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 involved in the development of ILD in SSc patients.

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

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. Based on these considerations, the hypotheses were made that (i) identified proteins may play a role in the onset and progression of UIP and OP and (ii) fragmented gelsolins may contribute to the development of pulmonary fibrosis.

and the multifunctional enzyme NDKB (an ion sensor in epithelial cells, pancreatic secretion, neutrophil-mediated inflammation, and energy production) were identified exclusively in the CF group. This interesting comparative study allowed the authors to identify deregulated proteins involved in tissue remodeling, complement system dysfunction with consequent impairment on defense mechanisms and chronic inflammation, nutritional imbalance, and

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

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

31

Chronic obstructive pulmonary disease (COPD) is an "umbrella" term that combines different pathological conditions such as emphysema, chronic bronchitis, nonreversible asthma, and some types of bronchiectasis characterized by irreversible airflow limitations. Its clinical features are a general progressive airflow limitation, destruction of the lung parenchyma, and/ or local fibrosis. Even though the main cause of the disorder is cigarette smoking, continuous inhalation of toxic gases and particles which promotes chronic airway inflammation may contribute to its development. COPD can also be determined by genetic factors, i.e., the deficiency

The following subsections describe electrokinetic-based proteomic studies aimed at elucidat-

To shed new light on the molecular mechanisms of COPD, Merali et al. [42] investigated the plasma proteome of 10 COPD patients and 10 healthy controls. Briefly, abundant proteins in pooled plasma from each group were immunodepleted; samples were then fractionated by 1-DE prior to ESI-MS/MS. A few proteins including acute-phase response proteins, CRP, fibrinogen, coagulation factors, and adhesion molecules were upregulated in COPD patients compared to controls. By contrast, molecules involved in protease-induced lung tissue injury

In a proteomic study aimed at identifying proteins involved in COPD pathogenesis, Ohlmeier et al. [43] analyzed induced sputum of nonsmokers, smokers, and smokers with moderate COPD by cysteine-specific 2D-DIGE coupled with MS. Among other candidates, polymeric immunoglobulin receptor (PIGR), a protein involved in specific immune defense and inflammation, appeared upregulated in smokers and subjects with COPD, thus suggesting its pos-

The application of 2-DE followed by LC-MS/MS allowed Plymoth et al. [44] to investigate the global proteome of BALf from 29 light and heavy smokers and 18 never-smokers in 6–7 years of follow-up study. During this study 7 of the 29 smokers developed moderate COPD.

sible role in the regulation of inflammation during COPD pathogenesis.

colonization by *P. aeruginosa*.

of α-1 antitrypsin (AATD).

and repair were downregulated.

**7.3. Bronchoalveolar lavage fluid**

**7.2. Induced sputum**

**7.1. Plasma**

**7. Chronic obstructive pulmonary disease**

ing the mechanism underlying the pathobiology of the disease.
