**4.2. Bronchoalveolar lavage fluid**

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

Asthma is an airway disease originating from complex interactions between genetic factors and environmental agents. It affects over 300 million people around the world and is characterized by airflow limitation resulting in shortness of breath. Bronchial obstruction is extremely variable and often reversible in asthma. Being a global health problem, tools which allow its early diagnosis, monitoring, and follow-up would offer great advantages for a better insight into its physiopathology. The articles discussed in the following sections prove that the proteomic content of sputum/induced sputum and BALf may allow for the identification of specific bio-

2-DE coupled to MALDI/TOF allowed Lee et al. [25] to compare the proteomic profiles of sputum from patients with neutrophilic-type uncontrolled asthma (UA) and from patients with neutrophilic controlled asthma (CA). It could be observed that, while a few proteins (including calgranulin S100A9) were overexpressed in sputum of UA patients, others, associated with inflammation, anti-inflammation, enzymatic activity, and immunity signaling, were downregulated. Differences in protein abundance and composition between asthma and rhinitis were revealed by the proteomic approach (2D-DIGE and MS) applied by Suojalehto et al. [26] to induced sputum (IS) and nasal lavage fluid (NLF) of patients. Their findings showed that (i) fatty acid-binding protein 5 (FABP5) was upregulated in IS of asthmatics, (ii) vascular endothelial growth factor (VEGF) was increased in NLF of asthmatics, and (iii) in NLF of these subjects,

B, C, and D, respectively).

26 Electrophoresis - Life Sciences Practical Applications

**4. Asthma**

markers in asthma.

**4.1. Sputum/induced sputum**

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 proteins included pulmonary surfactants and LPLUNC1.
