Quantitative Proteomics Research Of Nucleic Acid Binding Proteins And Serum And Their Application In Lung Cancer

Quantitative proteomics techniques are important tools for cancer research,especially for the discovery of therapeutic biomarkers and key regulatory proteins.The metastasis and drug resistance of lung cancer has always been a difficult problem in its treatment.How to evaluate the effect of cancer treatment requires the discovery of specific molecular indicators.As the most important body fluid of the human body,blood is a commonly used sample in clinical tests.Quantitative comparison of changes in serum proteome at different stages of lung cancer treatment can help to discover prognostic biomarkers for cancer treatment and explore the molecular mechanisms of cancer metastasis and drug resistance.Nucleic acid binding proteins(NABPs)widely mediate basic functions in cells.Due to the lack of efficient enrichment methods,their isolation and analysis are extremely challenging,and the development of new enrichment strategies will contribute to their functional research in cancer.In this study,a variety of quantitative proteomics techniques were used to systematically study NABPs and lung cancer serum proteome.The results are as follows.Firstly,a new NABPs enrichment strategy based on carbon nanomaterials was developed,and NABPs related to lung cancer progression were found.By comparing the differences in NABPs extraction of different carbon nanomaterials,we found that the hydroxyl and carboxyl groups carried by Graphene oxide(GO)could adsorb more nucleic acids through cation-mediated ionic bonds.From the GO-enriched samples,we successfully identified approximately 2,400 proteins.Eighty-eight percent of the identified NABPs were significantly enriched compared to the whole cell lysates,indicating the high efficiency and selectivity of the new NABPs extraction strategy.The developed method was used to compare NABPs in lung cancer cells with varying progression ability,and 117 differentially expressed NABPs were identified by labelfree quantitative proteomics.The validation results in lung cancer cells and lung cancer serum showed that the level of protein SAMHD1 was significantly increased in the progression of lung cancer,which can be used as a potential marker for the evaluation of lung cancer progression.Secondly,based on antibody microarrays,we discovered and validated a protein marker isoform 2 of fibrinogen alpha chain(FGA2),which was a prognostic indicator for the epidermal growth factor receptor(EGFR)mutated lung cancer targeted therapy.We used a microarray chip containing 41,472 antibodies to analyze serum samples from lung cancer patients with EGFR mutation at three time points: before targeted therapy,partial response after treatment(PR),and lung cancer progression after drug resistance(PD).After preliminary verification of the candidate antibody,the candidate protein was identified by immunoprecipitation combined with mass spectrometry,which led to the discovery of isoform 2 of fibrinogen alpha chain(FGA2).Validation in serum samples from the discovery and pre-validation groups showed that serum FGA2 level was correlated with EGFR tyrosin kinase inhibitor(TKI)targeted therapy(p < 0.05).The Area under curve(AUC)values of FGA2 in distinguishing between baseline group and normal group and PR group were 1.0 and 0.98,respectively.Cell line studies confirmed that EGFR-TKI can inhibit the expression and secretion of FGA2,partially revealing the mechanism of down-regulation of FGA2 in the serum of lung cancer patients with targeted therapy.Thirdly,through serum quantitative proteomics studies,we found that loss of TRIM33 promoted EGFR-TKI resistance in lung cancer by promoting the expression of SERPINE1.Using TMT and label-free quantitative proteomics techniques,EGFR-TKI resistance-associated proteins were identified in EGFR mutated lung cancer patients' serum and EGFR mutated lung cancer cell lines.After joint data analysis,three candidate proteins were identified and validated,including BZW1,TRIM33 and SERPINE1,which showed significant changes in the serum during targeted therapy,and were associated with the efficacy of targeted therapy(p < 0.05).Protein function studies have shown that the absence of TRIM33 could promote drug resistance in erlotinib-sensitive cell lines and enhance the invasive ability of lung cancer cells.We found that the expression of SERPINE1 was up-regulated in TRIM33 knockdown cells,and knockdown of SERPINE1 restored the sensitivity of lung cancer cells to EGFRTKI and decreased cell invasion ability.In summary,we focus on the metastasis and drug resistance of lung cancer.Based on quantitative proteomics,we developed new enrichment method for NABPs and conducted serum proteomics research,which resulted in the discovery of key proteins related to lung cancer metastasis and drug resistance.The new developed method of enriching NABPs has the advantages of simplicity,high efficiency,and high selectivity,which is expected to be widely used in biomedical research.FGA2 discovered by antibody microarray chip can be used as an indicator to evaluate the prognosis of EGFR mutated lung cancer targeted therapy,and provides an important reference for related research.The key proteins involved in drug resistance discovered and verified from serum samples have potential as targets for inhibiting drug resistance,and provide new ideas and strategies for elucidating cancer resistance mechanisms.