A novel method for sample preparation of fresh lung cancer tissue for proteomics analysis by tumor cell enrichment and removal of blood contaminants
In-depth proteomics analyses of tumors are frequently biased by the presence of blood components and stromal contamination, which leads to large experimental variation and decreases the proteome coverage. We have established a reproducible method to prepare freshly collected lung tumors for proteomics analysis, aiming at tumor cell enrichment and reduction of plasma protein contamination.
We obtained enriched tumor-cell suspensions (ETS) from six lung cancer cases (two adenocarcinomas, two squamous-cell carcinomas, two large-cell carcinomas) and from two normal lung samples. The cell content of resulting ETS was evaluated with immunocytological stainings and compared with the histologic pattern of the original specimens.
By means of a quantitative mass spectrometry-based method we evaluated the reproducibility of the sample preparation protocol and we assessed the proteome coverage by comparing lysates from ETS samples with the direct lysate of corresponding fresh-frozen samples.
Results: Cytological analyses on cytospin specimens showed that the percentage of tumoral cells in the ETS samples ranged from 20% to 70%. In the normal lung samples the percentage of epithelial cells was less then 10%.
The reproducibility of the sample preparation protocol was very good, with coefficient of variation at the peptide level and at the protein level of 13% and 7%, respectively. Proteomics analysis led to the identification of a significantly higher number of proteins in the ETS samples than in the FF samples (244 vs 109, respectively).
Albumin and hemoglobin were among the top 5 most abundant proteins identified in the FF samples, showing a high contamination with blood and plasma proteins, whereas ubiquitin and the mitochondrial ATP synthase 5A1 where among the top 5 most abundant proteins in the ETS samples.
Conclusion: The method is feasible and reproducible. We could obtain a fair enrichment of cells but the major benefit of the method was an effective removal of contaminants from red blood cells and plasma proteins resulting in larger proteome coverage compared to the direct lysis of frozen samples.
This sample preparation method may be successfully implemented for the discovery of lung cancer biomarkers on tissue samples using mass spectrometry-based proteomics.
Author: Luigi De PetrisMaria PernemalmGoran ElmbergerPer BergmanLotta OrreRolf LewensohnJanne Lehtio
Credits/Source: Proteome Science 2010, 8:9
Published on: 2010-02-26
Copyright by the authors listed above - made available via BioMedCentral (Open Access). Please
make sure to read our disclaimer prior to contacting 7thSpace Interactive. To contact our editors, visit our online helpdesk. If you wish submit your own press release, click here.
Social Bookmarking
RETWEET This! | Digg this! | Post to del.icio.us | Post to Furl | Add to Netscape | Add to Yahoo! | Rojo
|
|
