Proteomic analysis of human epithelial lining fluid by microfluidics‐based nanoLC‐MS/MS: A feasibility study

Abstract

Microfluidics‐based nanoLC‐MS/MS (chipLC‐MS/MS) was used to identify and quantify proteins in epithelial lining fluid (ELF), collected during bronchoscopy from the main bronchi of chronic obstructive pulmonary disease (COPD) patients and healthy controls using microprobes. ELF is a biofluid that is well suited to study pathophysiological processes in the lung, because it contains high concentrations of biologically active molecules. 1D‐PAGE followed by in‐gel tryptic digestion and chipLC‐MS/MS resulted in identification of approximately 300 proteins. A comparative study of ELF from COPD patients and non‐COPD controls using chemical stable isotope labeling (iTRAQ®‐8Plex) showed that the levels of lactotransferrin, high‐mobility group protein B1 (HMGB 1), alpha 1‐antichymotrypsin and cofilin‐1 differed significantly in ELF from COPD patients and non‐COPD controls (p‐values < 0.05). These results were reproduced in another, independent set of ELF samples from COPD patients and non‐COPD controls and further validated by immunohistochemistry. This study shows the feasibility of performing chipLC‐MS/MS and quantitative proteomics in human ELF.

Asthma and COPD proteomics: Current approaches and future directions

“…Finally, from the present overview of recent literature, new targeted approaches to the study of PTMs as well as quantitative labeling techniques seem to light up new important directions to explore. We expect that the ongoing development of methodological separation procedure coupled to MS platform and to more accurate quantitation and validation strategies, here exhaustively described, may offer reliable promise for the study of the yet not completely known complexity of asthma and COPD.…”

“…The only contradictory results were found for cofilin‐1, one of the differentially expressed proteins between COPD and non‐COPD controls. In fact, cofilin‐1 resulted increased in one set of ELF samples and decreased in a different set of ELF samples, both derived from COPD patients . However, the authors explained these contradictory results by reclassifying the subjects of the two parallel studies in smokers and nonsmokers, disregarding completely the eventual COPD pathology, and observing that cofilin‐1 (an actin depolymerizing factor) was significantly increased in ELF from smokers.…”

“…For example, Franciosi et al. found increased levels of expression for HMGB1 (an “alarmins” member, involved in the development and progression of acute lung injury), in ELF from non‐COPD individuals in comparison to COPD patients. These data were in conflict with those published by others using ELISA assays in BALF from COPD patients .…”

Searching for biomarkers of chronic obstructive pulmonary disease using proteomics: The current state

“…The use of ELF in the COPD area was mainly promoted by Franciosi's team to investigate the proteomics of healthy subjects and COPD patients.…”

“…To further evaluate the presence of putative COPD biomarkers in ELF, the same authors performed two comparative studies on a larger number of COPD and control samples (four samples/group) . They focused their attention on four proteins (lactotransferrin, α1‐anti‐chymotrypsin, cofilin‐1 and HMGB1) that were significantly over expressed in COPD patients compared to controls.…”