应用UPLC-QTOF-MS/MS技术测定肝细胞癌患者血清代谢组

Measurement of serum metabolomics of hepatocellular carcinoma patients using UPLC-QTOF-MS/MS technology

目的通过比较肝细胞癌(HCC)和肝硬化(LC)患者血清代谢物,研究HCC代谢谱变化,为寻找HCC早期诊断标志物及治疗新靶点和策略提供依据。方法收集HCC和LC患者血清各50例,应用超高效液相色谱-四极杆飞行时间质谱联用技术(UPLC-QTOF-MS/MS)对血清代谢物进行鉴定和定量。液相色谱分离采用反相色谱(RPLC)和亲水色谱(HILIC)2种模式,质谱采集包括正、负2种离子化方式。应用Progenesis QI软件进行数据处理,应用EZinfo软件进行主成分分析和正交偏最小二乘判别分析等多元统计分析并筛选差异代谢物。代谢物鉴定通过检索人代谢组数据库(HMDB)和Metlin数据库实现。应用MetaboAnalyst 4.0对差异代谢物进行代谢通路分析。结果应用UPLC-QTOF-MS/MS技术,共检测到87610个特征代谢峰,鉴定出7848种代谢物。通过P<0.05,变量重要性投影指标(VIP)值≥1及HCC与LC比较的差异倍数≥1.5或≤0.67等3个条件筛选出差异代谢物438个,主要包括磷脂、胆汁酸、游离脂肪酸、小肽和氨基酸等,涉及12个代谢通路的变化。结论与LC患者比较,HCC患者的血清代谢组发生显著变化,共有438种差异代谢物参与12个代谢通路的变化,这有助于发现早期诊断标志物和药物靶标。其中50个差异代谢物具有较高的临床诊断价值,值得进一步验证。另外,本研究近8000个代谢物的鉴定表明,所采用的RPLC结合HILIC分离的UPLC-QTOF-MS/MS技术显著提高了代谢物的鉴定覆盖率,可广泛应用于多领域的代谢组学研究。

OBJECTIVE To make a comparative study of serum metabolites of hepatocellular carcinoma(HCC)and liver cirrhosis(LC)and to investigate the metabolome alteration of HCC in order to help find potential diagnostic biomarkers and therapeutic targets for clinical treatments.METHODS The serum metabolites of 50 HCC and 50 LC patients were identified and quantified by ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry(UPLC-QTOF-MS/MS)techniques.The complementary separation by reversed phase liquid chromatography(RPLC)and hydrophilic interaction chromatography(HILIC)was carried out to increase the identification coverage of metabolites.Each sample was analyzed in both the positive and negative ionization mode.In total,four LC-MS runs were performed for each sample.Data related to peak alignment,peak picking and normalization was processed with Progenesis QI software.Principal component analysis(PCA),orthogonal partial least squares-discriminant analysis(OPLS-DA)and analysis of variance(ANOVA)were conducted using EZinfo software to characterize the significant differential metabolites.The metabolites were identified by searching Human Metabolome Database(HMDB,downloaded from www.hmdb.ca)and Metlin(purchased from Waters company)according to mass accuracy,isotope similarity and MS/MS spectra.RESULTS Using a non-targeted metabolomic strategy based on UPLC-QTOF-MS/MS,87610 metabolic features were detected and 7848 metabolites were identified by searching HMDB and Metlin database.Among the identified metabolites,438 significantly-changed ones were identified,which met the strict criteria of P<0.05,the variable importance in the projection(VIP)≥1 and fold change(FC)≥1.5 or≤0.67.The differential metabolites were mainly from phosphoplipids,bile acids,free fatty acids,peptides and amino acids.Pathway analysis indicated that the differential metabolites were involved in sphingolipid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,glycerophospholipid metabolism and alpha-linolenic acid metabolism pathways.CONCLUSION Significant alteration of HCC serum metabolomes occurs compared with LC.In total,438 differential metabolites have been quantified and the top 50 metabolites are of high value for early diagnosis and worthy of more validation.These metabolites may provide useful information for identification of early diagnostic biomarkers and therapeutic targets for HCC.In addition,the identification of nearly 8000 metabolites indicates that metabolic coverage can be increased greatly by using this UPLC-QTOF-MS/MS based technique combined with RPLC and HILIC separation,which can be used widely in various metabolic studies.