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Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer.

Liu Y, Luo X, Hu H, Wang R, Sun Y, Zeng R, Chen H - PLoS ONE (2012)

Bottom Line: This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood.Of these proteins, Alpha-1B-glycoprotein (A1BG) and Leucine-rich alpha-2-glycoprotein (LRG1), two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients.We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT
Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC) can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA) and Multiple reaction monitoring (MRM) assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG) and Leucine-rich alpha-2-glycoprotein (LRG1), two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

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Workflow of western blotting assisted verification for A1BG.Western blot images across 12 ADs, 12 SCCs and 12 control sera. Coomassie stained Albumin (ALB) was shown as the loading controls. IOD values were extracted and normalized by this calibration sample, suggesting the differential distribution of A1BG serum levels between normal and lung cancer cases.
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pone-0051748-g005: Workflow of western blotting assisted verification for A1BG.Western blot images across 12 ADs, 12 SCCs and 12 control sera. Coomassie stained Albumin (ALB) was shown as the loading controls. IOD values were extracted and normalized by this calibration sample, suggesting the differential distribution of A1BG serum levels between normal and lung cancer cases.

Mentions: Because of the antibody issues or the limited MRM sensitivity, we could not detect USP1 and muscin 5B in NSCLC serum samples, we then focus on the validation of A1BG and LRG1 in the following experiments. Western blotting (WB) validation was done only for A1BG (Figure 5) due to antibody compatibility. To find effective way to do WB for proteomic verification, i.e. to profile the protein expressions across a large sample scale after discovery phase by MS analysis, we recruited one “calibration sample” which pooled all the 18 MS samples in the discovery phase with equal constitutes. This pooled sample was identically treated with other samples in every run of WB, providing an internal standard to link different WB membranes. After final WB images acquired, an image software was used to extract the protein expression data by IOD value. The expression levels in each WB run thus could be normalized by this calibration sample and be compared, suggesting that A1BG indeed had a significant differential expression between NSCLC and control group (p = 0.0021).


Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer.

Liu Y, Luo X, Hu H, Wang R, Sun Y, Zeng R, Chen H - PLoS ONE (2012)

Workflow of western blotting assisted verification for A1BG.Western blot images across 12 ADs, 12 SCCs and 12 control sera. Coomassie stained Albumin (ALB) was shown as the loading controls. IOD values were extracted and normalized by this calibration sample, suggesting the differential distribution of A1BG serum levels between normal and lung cancer cases.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526638&req=5

pone-0051748-g005: Workflow of western blotting assisted verification for A1BG.Western blot images across 12 ADs, 12 SCCs and 12 control sera. Coomassie stained Albumin (ALB) was shown as the loading controls. IOD values were extracted and normalized by this calibration sample, suggesting the differential distribution of A1BG serum levels between normal and lung cancer cases.
Mentions: Because of the antibody issues or the limited MRM sensitivity, we could not detect USP1 and muscin 5B in NSCLC serum samples, we then focus on the validation of A1BG and LRG1 in the following experiments. Western blotting (WB) validation was done only for A1BG (Figure 5) due to antibody compatibility. To find effective way to do WB for proteomic verification, i.e. to profile the protein expressions across a large sample scale after discovery phase by MS analysis, we recruited one “calibration sample” which pooled all the 18 MS samples in the discovery phase with equal constitutes. This pooled sample was identically treated with other samples in every run of WB, providing an internal standard to link different WB membranes. After final WB images acquired, an image software was used to extract the protein expression data by IOD value. The expression levels in each WB run thus could be normalized by this calibration sample and be compared, suggesting that A1BG indeed had a significant differential expression between NSCLC and control group (p = 0.0021).

Bottom Line: This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood.Of these proteins, Alpha-1B-glycoprotein (A1BG) and Leucine-rich alpha-2-glycoprotein (LRG1), two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients.We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT
Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC) can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA) and Multiple reaction monitoring (MRM) assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG) and Leucine-rich alpha-2-glycoprotein (LRG1), two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

Show MeSH
Related in: MedlinePlus