Article pubs.acs.org/jpr
Development of Urinary Pseudotargeted LC-MS-Based Metabolomics Method and Its Application in Hepatocellular Carcinoma Biomarker Discovery Yaping Shao,†,# Bin Zhu,‡,# Ruiyin Zheng,§,# Xinjie Zhao,† Peiyuan Yin,† Xin Lu,† Binghua Jiao,§ Guowang Xu,*,† and Zhenzhen Yao*,§ †
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China ‡ The Second Department of Biliary Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 225 Changhai Road, Shanghai 200438, China § Department of Biochemistry & Molecular Biology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China S Supporting Information *
ABSTRACT: Hepatocellular carcinoma (HCC) is one of the pestilent malignancies leading to cancer-related death. Discovering effective biomarkers for HCC diagnosis is an urgent demand. To identify potential metabolite biomarkers, we developed a urinary pseudotargeted method based on liquid chromatography−hybrid triple quadrupole linear ion trap mass spectrometry (LC−QTRAP MS). Compared with nontargeted method, the pseudotargeted method can achieve better data quality, which benefits differential metabolites discovery. The established method was applied to cirrhosis (CIR) and HCC investigation. It was found that urinary nucleosides, bile acids, citric acid, and several amino acids were significantly changed in liver disease groups compared with the controls, featuring the dysregulation of purine metabolism, energy metabolism, and amino metabolism in liver diseases. Furthermore, some metabolites such as cyclic adenosine monophosphate, glutamine, and short- and mediumchain acylcarnitines were the differential metabolites of HCC and CIR. On the basis of binary logistic regression, butyrylcarnitine (carnitine C4:0) and hydantoin-5-propionic acid were defined as combinational markers to distinguish HCC from CIR. The area under curve was 0.786 and 0.773 for discovery stage and validation stage samples, respectively. These data show that the established pseudotargeted method is a complementary one of targeted and nontargeted methods for metabolomics study. KEYWORDS: pseudotargeted method, LC−MS, metabolomics, urine, cirrhosis, hepatocellular carcinoma
1. INTRODUCTION
Metabolome, the end product of life activities, is an aggregate of low-molecular-weight compounds participating in metabolism, maintaining regular physiological function and regulating growth and development of living body.5,6 Metabolomics study in the liver disease process has drawn much attention to disease pathology investigation7,8 and biomarker discovery.9,10 A nontargeted metabolic profiling of a tissue study revealed a holistic view of the metabolic features of HCC: glycolysis, gluconeogenesis, and β-oxidation were up-regulated while tricarboxylic acid cycle was down-regulated in HCC tissue compared with healthy tissues.11 Wang et al. elucidated that the invasion and metastasis of HCC were related to the alteration of glycolysis and the dysregulation of glycine and choline metabolism.7 Besides, biomarkers discovery is a mainstream issue in HCC study.10,12,13 It was revealed that bile acids were
The morbidity of hepatocellular carcinoma (HCC) has increased rapidly in recent years, and the mortality rate has been in third place in malignancy diseases.1,2 In China, most HCC develops from liver cirrhosis (CIR), which is primarily caused by chronic hepatitis B virus (HBV) or chronic hepatitis C virus (HCV) infection.3 The lack of early diagnosis and effective therapeutic protocol for advanced cancer are the most primary factors causing the high mortality rate. Because of the biological heterogeneity of liver diseases, it is difficult to distinguish HCC from other liver diseases such as CIR or fatty liver, simply by the clinical symptoms or pathophysiological characteristics.3 Currently, although serum α-fetoprotein (AFP) is a generally accepted biomarker for diagnosis of HCC in clinical, the sensitivity and specificity are not satisfactory.2,4 Hence there is an urgent demand to find new biomarkers of HCC, which can be used specifically and sensitively in diagnosis as well as in prognosis and therapy evaluation. © XXXX American Chemical Society
Received: September 13, 2014
A
dx.doi.org/10.1021/pr500973d | J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Article
Figure 1. Experimental scheme.
method has been exploited in disease study23 and plant investigation24 by using GC−MS as well as plasma metabolic profiling study by using UPLC−MS.18 Urine specimen is easily available and noninvasive to individuals; the preparation is simple as few proteins are contained, and hence it fits well for disease biomarker discovery.25 Nevertheless, a urinary pseudotargeted method based on LC−MS has not been reported. In this study, we developed a urinary pseudotargeted method based on UPLC−MS, and then the established pseudotargeted method was applied to liver diseases study. Our aim is to investigate the changes of urinary metabolic profiling of liver diseases and to find potential biomarkers for the discrimination of HCC and CIR. The experimental scheme is illustrated in Figure 1.
important biomarkers for HCC cases, in which elevated serum bile acids level was observed due to the injury of liver and dysregulation of enterohepatic circulation.9,14,15 A serum metabolomics study concentrated on HCC infected with HBV and HCV found that anandamide and palmitylethanolamide showed favorable sensitivity and specificity in distinguishing HCC from CIR infected with HCV.16 Dai et al. analyzed the urinary steroid hormone in early HCC patients, CIR patients, and healthy controls, in which epitestosterone and allotetrahydrocortisol were found to have a favorable capacity to distinguish HCC from CIR.12 Liquid chromatography−mass spectrometry (LC−MS) is a powerful approach for metabolomics study due to its simplified sample pretreatment, wide coverage of metabolites, and high sensitivity of mass spectrometry.17 Nontargeted method is a common employed approach in metabolomics study, which is able to detect as many metabolites as possible without previous knowledge of sample components.18,19 Nevertheless, there are still some deficiencies, such as complicated matrix influence,20 peak alignment default,21 and repeatability problem in largescale nontargeted measurement.18 Multiple reaction monitoring (MRM) mode is a key MS signal collection technology in the targeted method. The combination of precursor ions and product ions is used to monitor metabolites specifically, which improves the signal-to-noise ratio and reduces complicated data processing. To integrate the advantages of nontargeted and targeted methods, recently, we have proposed a new metabolomics method,18,22 in which ion pairs of metabolites are defined from the metabolic profiling of nontargeted analysis; then, based on these ion pairs the MRM mode is used to detect metabolome information on each sample to measure as many metabolites as possible that are similar to those in the nontargeted method. To distinguish it from common “targeted method”, we named our method the “pseudo-targeted method” because the metabolites in the MRM detection were not identified. It provides better repeatability and wider linear range.18 Furthermore, pseudotargeted method was performed by triple quadrupole MS, which was more common for most laboratories. Pseudotargeted
2. MATERIALS AND METHODS 2.1. Chemicals and Reagents
HPLC-grade solvent (acetonitrile, methanol) and formic acid were purchased from Merck (Germany) and Fluka (Germany), respectively. Ultrapure water used in the study was prepared by a Milli-Q system (Millipore, USA). Chemical standards for structure identification were obtained from Sigma-Aldrich (USA). The nine internal standards were supplied by Sigma-Aldrich (USA), including acetyl-d3-L-carnitine, decanoyl-d3-carnitine, hexanoyl-d3-carnitine, leucine-d3, phenylalanine-d5, tryptophand5, cholic acid-2,2,4,4-d4, chenodeoxycholic acid-2,2,4,4-d4, and leucine enkephalin. Then, internal standards were dissolved into methanol for urine metabolites extraction. Details of the concentration of internal standards are attached in Supplementary Table S1 in the Supporting Information. 2.2. Urinary Specimen Collection and Pretreatment
The study was approved by the Ethics Committee of Eastern Hepatobiliary Surgery Hospital, and each enrolled patient signed informed consents. In the discovery stage, 60 patients were involved, including 27 CIR subjects and 33 HCC subjects. In addition, 26 healthy individuals were recruited as a control B
dx.doi.org/10.1021/pr500973d | J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Article
Table 1. Clinical Information in Discovery Set and Validation Set discovery
validation
characteristics
HC
CIR
HCCa
CIR
HCCb
number sex (male/female) age AFP (μg/L)
26 21/5 53.2 ± 9.4
27 22/5 53.4 ± 9.6 54.6 ± 133.4
33 28/5 52.3 ± 10.3 443.3 ± 556.2c
21 16/5 49.2 ± 11.7 35.7 ± 95.3
33 27/6 57.6 ± 11.9 299.1 ± 523.3
a
In 33 HCC cases, 9 cases were small HCC patients (tumor size was
