**3. Principles of electrokinetic procedures**

branches of biochemistry. It is no exaggeration to say that, thanks to the significant step forward in the sensitivity and specificity of analytical methods, (almost) all proteins expressed by an organism can be detected/quantified even in extremely complex biological matrices. The monitoring of protein expression patterns in clinical specimens may indeed offer great opportunities to establish sets of biomarkers potentially associated with a specific disease status [1–8]. Despite these advantages, the question arises whether the application of these sophisticated proteomic procedures have profoundly affected the study of human diseases. If the number of publications in this field counts for something, the answer is undoubtedly positive. Through its ability to provide insights into both specific and system-level changes in cell, tissue and human physiology, proteomics has driven the progress observed in the last years in the elucidation of a variety of multifactorial pathological conditions, including less commonly diagnosed disorders [9–13]. In clinical proteomics several tissues and/or biological fluids are routinely analyzed for expressed proteins. Obviously, to make these analyses quantitative and reproducible, reliable profiling procedures should be established. Such procedures must rely on efficient and robust separation systems whose pivotal role is to make the complexity of mixtures simpler. Electrophoretic separations, both in-gel (1-DE and 2-DE) and in-solution (capillary electrophoresis (CE) and liquid chromatography (LC) approaches, both coupled to mass spectrometry (MS)), are currently the most attractive strategies toward the separation of hundreds/thousands of proteins. While acknowledging a remarkable success of electrophoretic approaches over the years, their intrinsic limitations cannot be hushed up. For example, the poor (if any) resolution of hydrophobic membrane proteins or of proteins that are too basic/acidic or too large/small is a well-known limit of 2-DE. The high protein amount required, the long time needed to run the samples, and the difficulty in automating the whole system are additional potential drawbacks of these techniques. The advent of LC-based procedures, characterized by unquestionable advantages, seemed to mark the fate of electrokinetic methods in the proteomic area. Being a bit of years elapsed, it can be argued that, despite the strong competition with LC, gel-based techniques do not appear to have lost this "struggle" yet [14–17]. Electrokinetic approaches, in fact, not only maintain their position but also still possess numerous characteristics currently unmatched by other proteomic methodologies. 2-DE approaches in fact remain the conventional procedure

applied to the differential (control *vs.* diseased case) analysis of biological samples [18–24].

The aim of this chapter is to illustrate the potential of electrokinetic procedures to the investigation of a variety of acute and chronic lung disorders. Why focus on pulmonary disorders? First, because lung diseases, which involve tens of million people, are some of the most common medical conditions in the world. Second, while few proteomic studies had been specifically designed to address this topic in the past, the depth of analysis ultimately reached by current procedures provides a new and larger context for future studies on the biology of these pathologies. This has allowed for the generation of protein profiles that are useful for exploring protein-based pathological mechanisms and/or discovering new potential thera-

Each paragraph of this chapter will address the proteomic data relative to a specific lung disease. Given that proteomic studies on pulmonary disorders have been carried out mostly on biofluids, the chapter will focus on the protein profiles obtained from serum/plasma, bronchoalveolar

**2. Outline of the chapter**

24 Electrophoresis - Life Sciences Practical Applications

peutic targets for a variety of pulmonary disorders.

Since electrokinetic approaches play a pivotal role throughout the whole chapter, a few details about the principles of techniques cited in the following paragraphs will be given here. The term "electrokinetics" refers to the motion of charged particles by an applied electrical field. The standard laboratory technique by which charged molecules migrate through a porous matrix which

**Figure 1.** Representative examples of 1-DE, 2-DE, 2D-DIGE, and CE: panels A, B, C, and D, respectively (unpublished results from our laboratory).

behaves like a molecular sieve is electrophoresis. Polyacrylamide is an ideal support for separating most proteins; under denaturing/reducing conditions and with a discontinuous buffer system, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or 1-DE is the most widely used electrophoretic technique separating proteins primarily by mass. When the ionic detergent SDS binds to proteins, they assume a uniform negative charge. Upon application of a current, all SDS-bound proteins in the sample will migrate toward the positive electrode, and, because of the sieving effect of the gel matrix, proteins with less mass split from those with greater mass because they travel more quickly. When the mixture of proteins is very complex, higher resolution may be obtained by applying a two-dimensional (2-DE) procedure which separates proteins according to their isoelectric point in the first dimension followed by an orthogonal separation via SDS-PAGE in the second one. Similar to 2-DE is two-dimensional difference gel electrophoresis (2D-DIGE) in which, however, two or more samples are labeled with different fluorescent dyes and separated on the same gel, thus eliminating gel-to-gel variability. An instrumental evolution of the abovementioned electrophoretic techniques is capillary electrophoresis (CE) in which separation occurs in fused-silica capillaries and separation of proteins involves application of high voltages across buffer-filled capillaries. Once separated by electrophoresis, gel bands/spots are excised and proteins extracted for analysis by mass spectrometry.

FABP5 and VEGF were positively correlated with cysteinyl leukotriene (CysLT). Based on their results, the authors hypothesized that, in asthma, FABP5 may contribute to the airway remodeling and inflammation by regulating the levels of CysLTs which, in turn, induce VEGF production.

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

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

27

A differential study on BALf of asthmatic individuals was performed by Wu et al. [27] who produced the first comprehensive database of BALf proteins. Abundant proteins were depleted by immunoaffinity chromatography, and all others were separated by 1-DE and identified by nano-LC-MS/MS. Chemokines and cytokines and a variety of matrix metalloproteases (MMPs) were upregulated in subjects after segmental allergen challenge. Other highly overexpressed

Interstitial lung disease (ILD) describes a collection of more than 200 lung disorders that involve inflammation and fibrosis of the alveoli, distal airways, and septal interstitium of the lungs. Typically, ILD presents with progressive breathlessness, lung crackles, and a diffusely abnormal chest radiograph. This disorder can be caused by long-term exposure to hazardous materials or by some types of autoimmune diseases, such as rheumatoid arthritis. In some cases, however, the etiology remains unknown. The following sections summarize the latest proteomic studies aimed at clarifying the pathophysiological mechanisms involved in the development and progression of ILD. All recent proteomic studies in this area based on electrokinetic approaches

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

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

use of an electrokinetic procedure have been performed on BALf.

**4.2. Bronchoalveolar lavage fluid**

**5. Interstitial lung disease**

have been performed on BALf.

**5.1. Idiopathic pulmonary fibrosis**

proteins included pulmonary surfactants and LPLUNC1.

Representative examples of 1-DE, 2-DE, 2D-DIGE, and CE are shown in **Figure 1** (panels A, B, C, and D, respectively).
