Using UHPLC Q-Trap/MS as a complementary technique to in-depth mine UPLC Q-TOF/MS data for identifying modified nucleosides in urine.
Modified nucleosides, metabolites of RNA, are potential biomarkers of cancer before the appearance of morphological abnormalities. It is of great significance to comprehensively detect and identify nucleosides in human urine for discovery of cancer biomarkers. However, the lower abundance, the greater polarity and the matrix effects make it difficult to detect urinary nucleosides. In this paper, an integrated method consisted of sample preparation followed by ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC Q-TOF/MS) detection and primary identification, then ultra-high performance liquid chromatography coupled with hybrid triple quadrupole linear ion trap mass spectrometer (UHPLC Q-Trap/MS) further identification and validation were introduced. Firstly, to enrich the nucleosides and eliminate the urine matrix effects, different sorbent materials of solid phase extraction (SPE) and the elution conditions were screened. Secondly, UPLC Q-TOF/MS was used to acquire mass data in MSEmode. The structural formulas of nucleosides in urine sample were primarily identified according to retention time, accurate mass precursor ions and fragment ions from in-house database and online database. Thirdly, the preliminary identified nucleoside structures lacking of characteristic fragment ions were verified by UHPLC Q-Trap/MS in multiple reaction monitoring trigger enhanced product ion scan (MRM-EPI) and neutral loss scan (NL). At last, phenylboronic acid (PBA)-based SPE was utilized due to its higher MS signal and weaker matrix effects under optimized extraction conditions. Fifty-five nucleosides were primarily identified by UPLC Q-TOF/MS, among which 50 nucleosides were confirmed by UHPLC Q-Trap/MS. Five nucleosides, namely 4',5'-didehydro-5'-deoxyadenosine, 4',5'-didehydro-5'-deoxyinosine, isonicotinamide riboside, peroxywybutosine and hydroxywybutosine, were found from urine for the first time. The results will expand the Human Metabolome Database (HMDB).
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: luzhiwei5233@sina.com.
Zhiwei
Z
Lu
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: wangqing_0046@bucm.edu.cn.
Qing
Q
Wang
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: wangmeiling@bucm.edu.cn.
Meiling
M
Wang
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: fushuang_5716@163.com.
Shuang
S
Fu
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: zhangqingqing1826@bucm.edu.cn.
Qingqing
Q
Zhang
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: zzxzxz@163.com.
Zhixin
Z
Zhang
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: zhaohuizhen_2013@126.com.
Huizhen
H
Zhao
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: liuyuehong@bucm.edu.cn.
Yuehong
Y
Liu
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: huangzhenghai0108@163.com.
Zhenhai
Z
Huang
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: xieziye200527@sina.com.
Ziye
Z
Xie
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: yuhonghong@bucm.edu.cn.
Honghong
H
Yu
School of Chinese Pharmacy, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing 100102, PR China. Electronic address: gaoxiaoyan0913@sina.com.
Xiaoyan
X
Gao
D000328:Adult; D001897:Boronic Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D009705:Nucleosides / Q000032:analysis / Q000302:isolation & purification / Q000652:urine*; D052616:Solid Phase Extraction / Q000379:methods*; D055815:Young Adult
D001897:Boronic Acids; D009705:Nucleosides; C010686:benzeneboronic acid
Phenylboronic acid; Solid phase extraction; Ultra-high performance liquid chromatography coupled with hybrid triple quadrupole linear ion trap mass spectrometer; Ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry; Urinary nucleosides