Human bronchoalveolar lavage: Biofluid analysis with special emphasis on sample preparation

Abstract

Respiratory diseases are an important health problem throughout the world. Whether caused by industrial pollutants, infections, smoking, cancer or metabolic diseases, damage to the lungs and airways often lead to morbidity or death. Bronchoalveolar lavage (BAL) obtained by fiber-optic bronchoscopy is a biofluid mirroring the expression of normally secreted pulmonary proteins and the products of activated cells and destructive processes. The characterization of the proteome within this compartment provides an opportunity to establish temporal and prognostic indicators of airway disease. The objective of this study was to develop methods of analysis of BAL samples, which achieved the highest level of annotation of the expression map of this proteome. We have optimized the process of sample preparation after investigating a variety of techniques including dialysis, ultramembrane filtration, precipitation and gel filtration. We have further studied methods to remove albumin from BAL in order to unmask proteins hidden on two-dimensional gels. In a pilot application of the method, BAL protein profiles obtained from healthy nonsmokers and smokers at risk for developing chronic obstructive pulmonary disease showed distinct differences.

Human body fluid proteome analysis

“…BALF is presently the most common way of sampling the components of the epithelial lining fluid and the most faithful reflect of the protein composition of the pulmonary airways. The characterization of the proteome within this compartment provides an opportunity to establish temporal and prognostic indicators of airway diseases [397,398].…”

“…The current master gel of BALF proteins comprises more than 1200 spots visualized by silver staining [402]. Identification of proteins on 2-D gels was initially performed using immunoblotting, Edman sequencing, or matching to a reference gel [403,404], and this was significantly improved through the use of advanced MS identification techniques [405][406][407][408][409]. A large-scale proteomic analysis by using immunoaffinity depletion, SDS-PAGE fractionation, protein in-gel digestion, and subsequent nano-LC-MS/MS was reported for identification and semiquantitation of more than 1500 distinct proteins in BALF.…”

Protein Expression Patterns Associated with Progression of Chronic Obstructive Pulmonary Disease in Bronchoalveolar Lavage of Smokers

“…BAL fluid containing 100 g protein was desalted by ultramembrane filtration and loaded using a pI 4 -7 immobilized pH gradient strip. We performed quantitative analysis with the image software PDQuest™ version 6.0 (Bio-Rad) (16,17 ).…”

“…We generated 2-D PAGE protein maps according to our previously developed procedure (16 ). BAL fluid containing 100 g protein was desalted by ultramembrane filtration and loaded using a pI 4 -7 immobilized pH gradient strip.…”

From Biomarker Discovery to Clinical Evaluation for Early Diagnosis of Lung Surgery-Induced Injury

“…In comparison, one-dimensional gel electrophoresis provides easy comparison of banding patterns in protein profiling but is less efficient in protein separation. Moreover, the high salt concentration in the bronchoalveolar lavage fluid interferes with the resolution of protein separation to a lesser extent in one-dimensional gel electrophoresis (Plymoth, 2003).…”