Identification Of Novel Candidate Sputum Metabolite Biomarkers For Distinguishing Lung Adenocarcinoma By ND-EESI-MS

Lung cancer is the most common malignant tumor with global morbidity and mortality.About 80% of lung cancer is Non-small cell lung cancer(NSCLC),with the change of lung cancer disease spectrum in recent years,the proportion of lung adenocarcinoma(LAC)patients in NSCLC has been risen to about over 50% compare less than 20%(decades ago).Improving the early diagnosis rate of lung adenocarcinoma is the key to improving the prognosis of lung cancer.Due to the lack of effective lung cancer screening and early diagnosis methods,about 85% of the currently diagnosed lung cancer is in the middle or late stage,which is an important cause of the poor overall 5-year survival rate of lung cancer.The research of the ideal screening method may be first important strategy to improve the early diagnosis rate and survival rate of lung cancer.Sputum is currently the only non-invasive diagnosis of lung cancer,and it has the advantages of non-invasive sampling,patient compliance,repeated sampling,and free X-ray exposure.Sputum is an ideal lung cancer screening and early diagnosis sample.Although the sputum examination has the best specificity for lung cancer diagnosis,the poor sensitivity limited its clinical value.Mass spectrometry has the characteristics of high sensitivity,good specificity and fast response.Mass spectrometry is the most sensitive method for determining molecular structure too.With self-developed neutral desorption electrospray ionization extraction mass spectrometry(ND-EESI-MS)method,this research is to study the application value and metabolic basis in the identification of lung cancer sputum by ND-EESI-MS.This study consists of the following two parts.1.Discrimination of lung cancer patients sputum by ND-EESI-MS.Objective: To explore the application value of neutral desorption electrospray ionization mass spectrometry(ND-EESI-MS)in identification of sputum from lung cancer.Methods: Neutral desorption electrospray ionization mass spectrometry (ND-EESI-MS)was used to detect sputum from lung cancer patients,lung benign disease patients and health examiners.The ND-EESI ion source was set to positive ion mode,the mass range was set to 50~500Da,the temperature of ion transmission tube was set to 150 V,the ionization voltage was 3.5kv,the lens voltage was 60 V,and the pressure of electrospray nitrogen gas was 0.4~0.6Mpa,the desorption nitrogen pressure was 3.2Mpa,and other conditions are automatically optimized.The PLS analysis was realized by programming in Matlab7.8,and the relative abundance of ions was statistically analyzed in SPSS 21.Results: Comparison showed that there was no significant difference between the benign lung disease and healthy.Coupling ND-EESI-MS and PLS analysis can clearly distinguish mass spectrometry fingerprint data of two groups samples,and ions of m/z238,m/z251,m/z274,m/z279,m/z348,m/z374,m/z379 and m/z391 have a greater contribution to the distinction between two groups samples.The relative abundance of m/z238,m/z279,m/z348 and m/z379 in sputum of lung cancer were significantly higher than the control group,while the ion m/z251 and m/z391 were higher in control group than lung cancer patients.Summary:: Coupling ND-EESI-MS and PLS analysis could identify the sputum of lung cancer,and might be a novel method for diagnosis and screening of lung cancer.2.Identification of novel candidate sputum metabolite biomarkers for distinguishing Lung Adenocarcinoma.Objective: To evaluate the potential of spontaneous sputum as a source of non-invasive metabolomic biomarkers for lung adenocarcinoma(LAC)diagnosis.Methods: Neutral desorption electrospray ionization mass spectrometry(ND-EESI-MS)was used to detect sputum from 62 cases of LAC and 60 cases of control group(39 cases of benign lung disease and 21 cases of healthy examination).The ND-EESI ion source was set to positive and negative ion mode,the mass range was set to 50~500Da,the temperature of ion transmission tube was set to 150 V,the ionization voltage was 3.5kv,the lens voltage was 60 V,and the pressure of electrospray nitrogen gas was 0.4~0.6Mpa,the desorption nitrogen pressure was3.2Mpa,and other conditions are automatically optimized.The relative abundance data was analyzed by PLS-DA in Simca,the mass-to-charge ratio(m/z)compound was considered to be novel candidate sputum metabolite biomarkers when model’s projection value(VIP> 1).The multi-stage collision-induced dissociation experiment(CID)was used to identify the VIP compounds.Metabo Analyst 4.0 software was performed to metabolic pathway analysis.Results: The relative abundances of ND-EESI-MS signals m/z 302.38、m/z 141.01 和m/z 165.09 etc.in LAC sputum were significantly different from control group(P<0.05).The metabolic difference in pathways such as sphingolipid metabolism,arginine proline metabolism,pyruvate metabolism,and linoleic acid metabolism have greater contribution to the distinction between two groups.For distinguishing LAC,the sensitivity of candidate sputum metabolite biomarkers is about 86.5% and the specificity is about 79.3%.Summary: Based on the differential metabolic compounds,ND-EESI-MS can effectively distinguished LAC sputum samples.The metabolic pathway changes are the theoretical basis for sputum metabolomics analysis for the LAC diagnosis.Conclusion:1,Combined ND-EESI-MS technology coupled with PLS analysis,non-small cell lung cancer sputum can be well identified from control by the difference of sputum mass spectrometry fingerprint signal.2.Alert of amino acid and phospholipid metabolism are the molecular basis of lung adenocarcinoma sputum mass diagnosis.3.Based on mass spectrometry analysis of differential metabolites in sputum,it provides a new method of early diagnosis or screening of lung adenocarcinoma.