**10. General considerations**

recruiting cohorts of patients whose size allows detection of statistically significant data. Nevertheless, Chang et al. [54] and Sixt et al. [55] investigated the proteomics of ARDS; proteomic data on HAPE have been published by Ahmad et al. [56, 57] and by Yang et al. [58],

With more than 10.4 million new cases and about 1.8 million deaths each year, tuberculosis (TB) still remains an urgent global health problem. Being all deaths mainly due to the increasing spread of *Mycobacterium tuberculosis* drug-resistant strains, early diagnosis and treatment of infection would be essential for the prevention. However, routine laboratory tests for drugresistant TB with sufficient sensitivity and specificity are still not available. This is the rationale for the development of electrophoretic-based proteomic approaches aimed at identifying host TB-associated proteins or antigens useful for the serodiagnosis of drug-resistant *M. tuberculosis* strains. To better understand and monitor the disease process, Tanaka et al. [60] analyzed whole blood supernatants from TB patients by 2D-DIGE followed by MS. Among others, the authors observed that retinol-binding protein 4 (RBP4) and fetuin-A were significantly lowered in patients with active TB compared to controls, thus suggesting that they could be considered potential biomarkers for monitoring the course of the disease during clinical treatment.

**Method Advantages/disadvantages Lung disease Matrix Ref. no.**

Asthma BALf [27] IPF BALf/IS [29] CF IS [40] COPD Plasma [29, 42]

Asthma IS [25] IPF BALf [29–34] Sarcoidosis BALf [32, 35–36]

RA-ILD BALf [38] PLCH BALf [32, 39] CF Serum [41]

COPD BALf [29, 30, 44,

IPAH Serum [48–50] TB Serum [61]

BALf [32, 37]

46, 47]

Lung fibrosis associated to SSc

1-DE —Allows separation of all types of proteins, even those

—Often needs the coupling of another detection

technique, i.e., immunoblotting or MS

digestion, and peptide analysis by MS

2-DE —Good resolution of protein mixtures

—Overlapping of closely spaced bands leading to limited

—Allows discernment of posttranslational modifications —Comparison of multiple gels facilitated by image

—Unable to resolve low molecular weight proteins

—Presence of high-abundance protein (i.e., albumin, immunoglobulin) hiding low-abundance proteins —Final identification requires spot removal from gels,

insoluble in water

analysis software

—Low throughput

(<10 kDa)

resolution

and preliminary data on IPA have been produced by Brasier et al. [59].

**9. Tuberculosis**

34 Electrophoresis - Life Sciences Practical Applications

Although the characterization of the full proteome is still challenging, the recent technological innovations have improved our ability to obtain cross-sectional time and space snapshots of protein levels that reflect observed phenotypes more closely than those of genomic techniques. The current successes in the use of proteomic approaches to understand disease and enable drug development resulted in optimism that many more effective diagnostic tests and treatments tailored to genetic, environmental, and lifestyle factors of individuals will be developed. Showing a great ability in providing reference data for the identification of groups of individuals who share various attributes, these approaches have opened new opportunities for the discovery of potential diagnostic/prognostic protein biomarkers in several pulmonary disorders. By helping researchers in understanding the pathogenesis of respiratory diseases and improve patient care, the recent findings have indeed progressively increased the interest for the application of proteomics in clinical practice. The current chapter was designed to keep the reader informed about the present status of pulmonary proteome. Taken together, the results documented here demonstrate that, after a decade of activity, proteomics of pulmonary diseases is catching up with its promise. The constantly growing number of reports in this area supports the view of this approach as one of the decisive methodological tools for the identification/characterization of disease-associated proteins. In terms of experimental procedures, the basic options available for proteomic investigations consist in the identification of proteins through the use of gel-based or gel-free techniques followed by MS. Undoubtedly, the striking improvement in technologies related to accuracy, when coupled to quantitative approaches, has a great impact on the quality of the results. Obviously, the question arises of whether sophisticated technologies (such as the non-gelbased proteomic procedures) may actually be more fruitful, in terms of candidate protein marker identification, than "conventional" (read electrokinetic) approaches. In light of the versatility and high degree of reproducibility shown by these new potent strategies, a positive answer is perhaps not surprising, at least for one reason. The very high number of peptides identified and quantified results in a higher accuracy, which translates into improved alignment and quantification across spectra. Nevertheless, as documented in this chapter, despite being less sophisticated than competing ones, gel-based techniques still represent a widely used procedure able to generate a reliable protein "fingerprint." Though it may seem nonsense, it is precisely the "limited" amount of information produced by electrokinetic approaches that may result in an easy interpretation of data. The possibility to compare a sample in physiological and pathological conditions allows, in fact, immediate detection of possible relevant changes in protein expression which differentiate the two conditions. These changes are essential in demonstrating progression from health to disease and understanding the relationship between function and modification. The wide spectrum of examples presented in this chapter confirms that the application of 1-DE/2-DE/2-DIGE/CE (followed by MS) to a variety of biological fluids from individuals with different respiratory diseases may result in the production of data with clinical relevance which allow a better understanding of the molecular basis of the disorder investigated. However, as it can be observed from the data presented in this chapter, while peculiar proteins are pointed out as potential biomarkers of specific disorders, a good number of proteins is implicated across a variety of different diseases. This makes the notion of a single biomarker to indicate a specific disease more difficult. For example, while α2-macroglobulin and surfactant protein A have been indicated as candidate biomarkers of both lung fibrosis associated with systemic sclerosis and asthma [27, 37], the former protein (together with other proteins) was suggested to be also a potential biomarker of pulmonary embolism [52]. Indeed, for greater confidence in disease diagnosis or prognosis, a suite of biomarkers would provide more specificity than a single one. In other words, should the identification of hundreds of candidate biomarkers come at the

price of sacrificing their specificity? This discrepancy, however, is only apparent and may be reconciled with a harmonization of results. In fact, given that a single molecule can hardly discriminate complex processes without context, the finding of common signatures for several pulmonary disorders contributes to drawing intriguing parallels among them. This obviously results in a better understanding of the disease mechanism. On the other hand, the fact that several proteins found (e.g., in COPD) have not been reported in other chronic lung diseases suggests that the merits of proteomics in hunting down biomarkers with high specificity cannot be under-evaluated. In this context, the finding of significantly altered levels of cathepsin B, ATP synthase, and chaperonin in the BALf of female COPD patients, but not in that of males, while supporting the hypothesis that these proteins were the most prominent marker candidates for this gender only, confirmed the abovementioned merits of proteomics [45]. Proteomic studies have also discovered panels/clusters of oxidant/antioxidant enzymes that may be utilized for the assessment of disease severity [44]. Overproduction of ROS can also cause oxidative modifications of several important antioxidant/defense enzymes, which may be associated with alterations in enzyme conformation, and thus they can function as markers of the degree of oxidative stress present in the airways [46, 47]. The huge amount of experimental data generated in some cases (i.e., COPD and asthma) represent the first attempts to identify the principal pathways involved in the pathogenesis and already allow

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

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

37

As the goal of the authors is to offer a broad picture of the subject, readers interested in learning more about specific techniques or their application to pulmonary diseases are encouraged to refer to excellent review articles in this field which show the merits of proteomic techniques in producing qualitative and quantitative information on the protein patterns of a variety of human fluids/tissues [62–65]. Taken together, these articles represent a good resource which describes in depth the status of electrokinetic (and chromatographic) proteomic methods and

Aside from the interest in deciphering the function of individual proteins, the set of data produced by proteomic methods represent the starting point for studying large-scale interactions that serve to discover general important properties for interaction participation. The fact that highly interactive proteins are often well conserved and/or essential or that homologous proteins, and, in particular, proteins with domains from the same family, tend to interact more frequently than others will likely improve the knowledge of their intrinsic properties. Thus, the understanding of the role these proteins play in the pathogenesis of respiratory diseases, while opening the door to much more powerful protein diagnostics, reinforces the linkage between basic medical research and clinical laboratory medicine. Addressing these concerns is obviously a top priority for the field, the ultimate goal of researchers being to understand

There is no doubt that this branch of respiratory proteomics will have substantial improve-

provide a comprehensive picture of proteomics of pulmonary disorders to date.

the biology of disease and to translate this knowledge into the clinic.

interesting candidate biomarkers to emerge [26, 43].

**11. Conclusions**

ment in the future.

price of sacrificing their specificity? This discrepancy, however, is only apparent and may be reconciled with a harmonization of results. In fact, given that a single molecule can hardly discriminate complex processes without context, the finding of common signatures for several pulmonary disorders contributes to drawing intriguing parallels among them. This obviously results in a better understanding of the disease mechanism. On the other hand, the fact that several proteins found (e.g., in COPD) have not been reported in other chronic lung diseases suggests that the merits of proteomics in hunting down biomarkers with high specificity cannot be under-evaluated. In this context, the finding of significantly altered levels of cathepsin B, ATP synthase, and chaperonin in the BALf of female COPD patients, but not in that of males, while supporting the hypothesis that these proteins were the most prominent marker candidates for this gender only, confirmed the abovementioned merits of proteomics [45]. Proteomic studies have also discovered panels/clusters of oxidant/antioxidant enzymes that may be utilized for the assessment of disease severity [44]. Overproduction of ROS can also cause oxidative modifications of several important antioxidant/defense enzymes, which may be associated with alterations in enzyme conformation, and thus they can function as markers of the degree of oxidative stress present in the airways [46, 47]. The huge amount of experimental data generated in some cases (i.e., COPD and asthma) represent the first attempts to identify the principal pathways involved in the pathogenesis and already allow interesting candidate biomarkers to emerge [26, 43].

As the goal of the authors is to offer a broad picture of the subject, readers interested in learning more about specific techniques or their application to pulmonary diseases are encouraged to refer to excellent review articles in this field which show the merits of proteomic techniques in producing qualitative and quantitative information on the protein patterns of a variety of human fluids/tissues [62–65]. Taken together, these articles represent a good resource which describes in depth the status of electrokinetic (and chromatographic) proteomic methods and provide a comprehensive picture of proteomics of pulmonary disorders to date.
