Editorial
Development of an in-House Software Platform and its Application in Untargeted Ultraperformance Liquid Chromatography Coupled to Time-of-Flight Mass Spectrometry Analysis in Metabolomics and Traditional Chinese Medicine Studies
Liang Q*
1Department of pharmacology, Beijing Institute of Radiation Medicine, China
*Corresponding author: Qiande Liang, Department of pharmacology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijng, P. R. China
Published: 31 Aug, 2016
Cite this article as: Liang Q. Development of an in-House Software Platform and its Application in Untargeted Ultraperformance Liquid Chromatography Coupled to Time-of- Flight Mass Spectrometry Analysis in Metabolomics and Traditional Chinese Medicine Studies. Clin Surg. 2016; 1: 1104.
Editorial
The recent development in high-resolution mass spectrometry (MS) has afforded powerful
analytical equipments, e.g., ultra performance liquid chromatography coupled to time-offlight
mass spectrometry (UPLC TOFMS), for rapid qualitative analysis of samples as complex
mixtures in biomedical research. This is especially beneficial for the research fields as untargeted
metabolomics and pharmaceutical analysis of natural product, e.g., the traditional Chinese
medicine. However, a significant bottleneck in deriving biological knowledge from the studies is
the process of identification of the samples’ components, which is a labor-intensive step that follows
data acquisition and analysis and must occur before biological interpretation is possible [1]. To
address this bottleneck, several computer software platforms have been developed in recent years
for automated data processing [1 and 2]. In our laboratory, we developed our own software platform in
2012, the operation of which is very simple [3]. Unlike the reported software platforms that address
raw LC-MS data and require the Taverna or R environment, our software platform deals only with
the dataset of LC-MS features produced after the pre-processing step and requires nothing more
than a Microsoft Windows operating system and Microsoft Excel software. One component of our
platform is a software named “Searcher”, which works as an Excel “add-in” component and can make
automatic putative batch identifications by matching the measured m/z data list with a reference
m/z list. Another component is a series of in-house, localized, Excel-format databases that contains
accurate theoretical m/z values for multiple types of ions commonly observed in the electrospray
ionization (ESI). Currently the series includes a HMDB (Human Metabolome Database, http://www.
hmdb.ca/[4-6])-derived database (HMDBDDB), a database of observed metabolite signals in UPLC
TOFMS analysis of serum of Sprague-Dawley (SDRSDB), a database of observed metabolite signals
in UPLC TOFMS analysis of urine of Sprague-Dawley (SDRUDB) and a literature-derived chemical
database of the plants of Rehmannia, Angelica, Paeonia and Ligusticum (RAPLDB). The SDRSDB
and SDRUDB can be used for distinguishing the signals of endogenous metabolites from exogenous
metabolites in serum or urine samples of SD rats. The RAPLDB can be used to detect the signals of
reported phytochemicals of the four medicinal plant families. With these two components (i.e., the
Searcher software and localized database series), rapid automatic putative batch identification of
high-resolution LC-MS data can be achieved with very simple operations, and the obstacle with data
analysis in our laboratory was successfully overcome. Below are examples of some research works
in our lab using this platform.
Research 1, “Rapid comparison of metabolites in humans and rats of different sexes using
untargeted ultra performance liquid chromatography coupled to time-of-flight mass spectrometry
and an in-house software platform” [7]. In this study, untargeted UPLC TOFMS and our platform
(Searcher software plus HMDBDDB) were used for a rapid comparison of sex differences in urinary
metabolites in humans and in urinary and serum metabolites in Sprague Dawley (SD) rats. In
addition, the species differences of urinary metabolites between humans and SD rats were also
observed. Principle component analysis showed that all the observed metabolite sex differences
were more distinct in SD rats than in humans, indicating that the sex differences of human urinary
metabolites is small compared with that of SD rats. In SD rats, the observed metabolite sex differences
were more distinct in urine than in serum, indicating the importance
of urine analysis for metabolomics studies. The species differences
in the urinary metabolites of humans and SD rats were much more
distinct than any of the observed sex differences. Many sex- and
species-related markers were discovered and putatively identified. In
both humans and SD rats, steroid metabolites appeared to constitute
a major sex difference in urinary metabolites. This provides new proof
of the special importance of steroid metabolites in sex differences
from an untargeted metabolomics investigation, which is rare for
sex differences. Contrary patterns involving adrenocortical activity
appeared to exist between rodents and humans, which agrees with
previous reports. In the serum metabolites of SD rats, sex differences
in ascorbic acid or its isomer and pantothenic acid or its isomer, but
not in steroid metabolites, were prominent. Human-specific a-Nphenylacetyl-
l-glutamine and androsterone glucuronide were among
the putative identities of the markers discriminating humans and SD
rats. This study demonstrated the feasibility of our in-house software
platform and provides metabolite-related information on sex and
species differences.
Research 2, “Hepatotoxicity and nephrotoxicity assessment
of ethanol extract from Fructus Psoraleae in Sprague-Dawley rats
using a UPLC-Q-TOF-MS analysis of serum metabonomics” [8].
The purpose of this study was to investigate the toxic effect in rats
treated with the ethanol extract from FP (EEFP) and to explore the
underlying toxic mechanisms using a metabonomics approach.
SD male rats were randomly divided into four groups (n = 6).
Dosages were administered once daily for two continuous weeks.
Serum was analyzed by UPLC TOFMS. PCA and partial leastsquares
discriminate analysis (PLS-DA) models were built to show
the difference. Potential biomarkers were screened from S-plots
constructed following analysis with orthogonal partial least squares
discriminant analysis (OPLS-DA) and identified based on the
accurate mass and MSE information obtained from UPLC TOFMS
analysis. Compared with control rats, the hematological (white blood
cell (WBC), neutrophilic granulocytes (NEUT), monocytes (MON)
and biochemical (alanine transaminase (ALT), total bilirubin (TBIL),
creatinine (CRE) parameters were significantly increased (p< 0.05,
p< 0.01), liver and kidney showed mild injury in the EEFP 1.62 g/
kg group. PCA and PLS-DA enabled four clusters to be visualized.
Using our platform (Searcher software plus HMDBDDB and
SDRSDB), ten potential biomarkers contributing to the clusters
were identified, which were lysophosphatidylcholine LysoPC (20:4),
p-Cresol, ascorbic acid, p-Cresol sulfate, inosine diphosphate
(IDP), phosphatidylcholine (PC (14:1/20:5), PC (14:1/16:1),
PC (22:2/18:1), phosphatidylethanolamine PE (22:1/18:1), and
phosphatidylglycerolphosphate PGP (18:1/22:6). Those endogenous
metabolites were chiefly involved in phospholipid metabolism, amino
acid metabolism, purine metabolism and the antioxidant system. The
change of hematological parameters, biochemical parameters and
plasma metabolic pattern show that long term EEFP exposure at high
dose could induce liver and kidney toxicity in rats. Some potential
biomarkers like LysoPC, p-Cresol, ascorbic acid, p-Cresol sulfate and
PC have been found to be reasonable in explaining the toxic effects
mechanism of EEFP in rats. The work shows that the metabonomics
method is a promising tool in the toxic mechanism research of
traditional Chinese medicines.
Research 3, “Cardiotoxicity study of Shenfu compatibility in
rats based on metabonomics” [9]. This research was to research
the effect of Ginseng Radix et Rhizoma and Aconiti Lateralis Radix
Praeparata combination on cardiac toxicity in rats by UPLC TOFMS,
and explore the endogenous markers and molecular mechanism.
Data was analyzed using PCA, partial least-squares analysis and
our platform (Searcher software plus HMDBDDB and SDRUDB).
Decoctions of different combinations of Shenfu were given to male
Wistar rats at a dosage of 20g· kg (-1) for 7 days. Serum samples
were collected and analyzed to discover the endogenous metabolites
affected by drug administration. Results showed that contents of
glutathione, phosphatidylcholine and citric acid decreased in the
“mixed-decoction” group, while ascorbic acid, uric acid, D-galactose,
tryptophan, L-phenylalanine increased. The Shenfu “co-decoction”
group showed a similar or weaker trend as compared with the control
group, but mostly not significant statistically.
Research 4, “Chemical comparison between decoctions of Radix
Paeoniae Rubra and Radix Paeoniae Alba by UPLC-QTOF-MS”
[10]. This research employed the RAPLDB database. To investigate
the chemical difference between decoctions of Radix Paeoniae Rubra
and Radix Paeoniae Alba, samples from legal market were compared
using UPLC TOFMS. Data were statistically analyzed by PCA and
OPLS-DA. Constituents identification methods include comparing
with reference compounds, accurate m/z value analysis of TOFMS
data based on our platform (Searcher plus RAPLDB) for automatic
tentative identification, and QTOF-MS/MS fragment analysis. The
results indicate that oxypaeoniflorin and albiflorin are the most
important differential constituents in the decoctions of Radix Paeoniae
Rubra and Radix Paeoniae Alba, respectively. Moreover, content of
compounds such as 8-debenzoylpaeoniflorin, 1’-O-benzoylsucrose,
Mudanpioside F, Mudanpioside C/benzoloxypaeoniflorin,
Mudanpioside E, (1S,2S,4R)-trans-2-hydroxy-1,8-cineole-B-Dglucopyranoside,
saccharose, galloylpaeoniflorin/galloylalbiflorin
may also be of significant difference between the two kinds of
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