Abstract
Aim: To discover proteins that change in abundance in ELF (Epithelial Lining Fluid) from COPD patients versus healthy controls using a quantitative proteomics approach.
Methods: The ELF proteome from COPD patients and healthy controls was studied by 1D polyacrylamide gel electrophoresis followed by in-gel tryptic digestion to assess the feasibility of such an approach. 40 gel slices were obtained from each lane of the gel (corresponding to one patient). Digested samples were analyzed by nanoChip-LC-MS/MS using an ion trap.
We performed a quantitative pilot study of ELF from 4 COPD patients and 4 healthy controls [table 1] to test for statistically significant differences in protein levels. ELF samples were digested by trypsin, labeled with stable isotope-containing reagents (iTRAQ®, 8-plex) and processed by strong cation-exchange chromatography followed by nanoLC-MS/MS. In order to validate the results, a second quantitative analysis of an independent sample set (4 COPD vs 4 healthy) using the same methodological approach was done.
Results: The 1D electrophoretic approach resulted in more than 300 identified proteins. Most of the identified proteins were present in both COPD and healthy samples, although some proteins were only identified either in healthy control or in COPD samples.
The quantitative studies showed a number of proteins significantly different between ELF of COPD patients and controls, including 4 up-regulated proteins in common in both studies.
Conclusions: The obtained results show the possibility to discover proteins differentially expressed in ELF of COPD patients and controls. We are currently validating these proteins by western blot and immunohistochemistry.
- © 2011 ERS