Chemico-Biological Interactions 215 (2014) 7–16

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Mini-review

UPLC–MSE application in disease biomarker discovery: The discoveries in

proteomics to metabolomics
Ying-Yong Zhao a,⇑, Rui-Chao Lin b,⇑
a
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, The College of Life Sciences, Northwest University,
No. 229 Taibai North Road, Xi’an, Shaanxi 710069, PR China
b
School of Chinese Materia Medica, Beijing University of Chinese Medicine, No. 11 North Third Ring Road, Beijing 100029, PR China

a r t i c l e i n f o a b s t r a c t

Article history: In the last decade, proteomics and metabolomics have contributed substantially to our understanding of
Received 17 November 2013 different diseases. Proteomics and metabolomics aims to comprehensively identify proteins and metab-
Received in revised form 14 February 2014 olites to gain insight into the cellular signaling pathways underlying disease and to discover novel bio-
Accepted 28 February 2014
markers for screening, early detection and diagnosis, as well as for determining prognoses and
Available online 12 March 2014
predicting responses to specific treatments. For comprehensive analysis of cellular proteins and metab-
olites, analytical methods of wider dynamic range higher resolution and good sensitivity are required.
Keywords:
Ultra performance liquid chromatography–mass spectrometryElevated Energy (UPLC–MSE) is currently one
Proteomics
Metabolomics
of the most versatile techniques. UPLC–MSE is an established technology in proteomics studies and is
Ultra performance liquid chromatography now expanding into metabolite research. MSE was used for simultaneous acquisition of precursor ion
Mass spectrometry information and fragment ion data at low and high collision energy in one analytical run, providing sim-
MSE ilar information to conventional MS2. In this review, UPLC–MSE application in proteomics and metabolo-
Disease biomarker mics was highlighted to assess protein and metabolite changes in different diseases, including cancer,
neuropsychiatric pharmacology studies from clinical trials and animal models. In addition, the future
prospects for complete proteomics and metabolomics are discussed.
Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3. Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4. Ultra performance liquid chromatography–mass spectrometryElevated Energy (UPLC–MSE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5. UPLC–MSE-based proteomics application in disease biomarker discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.1. UPLC–MSE-based proteomics in clinical research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.2. LC–MSE-based proteomics in animal model or cell model research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6. Metabolomics application in disease biomarker discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
6.1. UPLC–MSE-based metabolomics in clinical research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
6.2. UPLC–MSE-based metabolomics in animal model research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
7. Conclusion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

⇑ Tel.: +86 29 88304569; fax: +86 29 88304368 (Y.-Y. Zhao). Tel.: +86 10 84738652; fax: +86 10 84738653 (R.-C. Lin).
E-mail addresses: zyy@nwu.edu.cn, zhaoyybr@163.com (Y.-Y. Zhao), linrch307@sina.com (R.-C. Lin).

http://dx.doi.org/10.1016/j.cbi.2014.02.014
0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
8 Y.-Y. Zhao, R.-C. Lin / Chemico-Biological Interactions 215 (2014) 7–16
1. Introduction
Diseases are often discovered in an advanced stage because of
the lack of high sensitivity and specificity biomarkers. An early
diagnosis is therefore of vital importance in order to increase the
survival rate, so novel biomarkers are urgently needed to stratify
patients and personalize treatments. Specific biomarker discovery
can be used to improve the accuracy of the clinical diagnosis. The
process of biomarker discovery involves analysis of biomarkers in
clinical patients or animal models. Systems biology including
genomics, transcriptomics, proteomics and metabolomics offers
enormous potential to understand the complexity of diseases.
Genomics, transcriptomics, proteomics and metabolomics are re-
lated to the genome (DNA), the transcriptome (RNA), proteome
(proteins) and metabolome (metabolites), respectively (Fig. 1).
Currently, biomarker assessment is based on the quantification of
a few proteins or metabolites [1]. Proteomics and metabolomics
have an important effect on disease studies because of their unique
strengths and because of the potential central pathogenic contribu-
tion of pathological proteins or metabolites to diseases. High
throughput platforms such as proteomics and metabolomics can
offer simultaneous readouts of hundreds of proteins and metabo-
lites. In this review, we summarize the UPLC–MSE-based proteo-
mics and metabolomics platforms that are currently applied in
disease research and that may lead to the identification of novel
biomarkers with clinical utility.
2. Proteomics
The Human Proteome Organization emerged from the Human
Genome Project as a means of understanding gene and protein
functions that may lead to the understanding of diseases and
to the identification of diagnostic/prognostic biomarkers. Since
proteins are responsible for all biological processes, changes in
the concentration and structures are likely to reflect disease
change, thereby making proteins attractive candidates in bio-
marker discovery. Proteomics is an emerging discipline for the
multivariate assessment of protein expression in biological sam-
ples and the possible comprehension of complex pathological
and physiological events using various techniques to identify
and characterize proteins. There has been a growing interest in
applying proteomics to research on clinical diagnostics and pre-
dictive medicine. Proteins types and concentrations can be fol-
lowed at set time using proteomics in biomarker discovery and
proteome correlation present in a disease state as compared to
healthy state can be of high diagnostic value to understand the
underlying disease mechanisms. The specific-proteins can aid
medicine in earlier diagnosis and treatment disease. Because dis-
ease often will involve various protein expressions, a combina-
tion of several biomarkers is generally more effective than a
single one.
Fig. 1. Schematic representation of omics technologies. The flow of information starts from genes to metabolites running through transcripts and proteins.
3. Metabolomics
Metabolomics is defined as the ‘‘quantitative measurement of
the dynamic multi-parametric metabolic responses of living sys-
tems to pathophysiological stimuli or genetic modifications’’ [2].
Metabolomics is used to characterize the biochemical patterns of
the endogenous metabolic compounds of serum, plasma, urine
and tissue. In contrast to traditional biochemical approach that often
focuses on a single metabolite, metabolomics is the analysis of col-
lection small molecules that are found within a system. It covers a
broad range of small molecules such as cholesterol, lipids, peptides,
amino acids, nucleic acids, carbohydrates, organic acids and vita-
mins tries to gain an overall understanding of metabolism and met-
abolic dynamics related to conditions of disease and drug exposure.
As a basis of medical research, small molecule research is now
reemerging from the limitations of molecular genetics, genomics,
proteomics and other fields that bring with them technologies of
immense power and insight. With the rapid development of
metabonomic platforms, it is now possible to more completely
visualize living organisms; the limited small molecules makes this
an easier, more quantitative approach of analysis and answers
pivotal problems that could not be fully addressed by the other
‘‘-omics’’ alone [2]. As a powerful analytical platform, the applica-
tion of metabolomics has remarkably increased in disease diagno-
sis, drug discovery, drug safety assessment and epidemiology.
From bacteria to humans, examples of this principle are accruing
at a rapid pace that has been made possible by remarkable recent
developments in analytical chemistry.
4. Ultra performance liquid chromatography–mass
spectrometryElevated Energy (UPLC–MSE)
The first proteomic techniques were developed in the 1970s.
Initially, Edman sequencing was used but the major hurdle was
the identification of proteins. This technique has been replaced
by the biological mass spectrometry (MS). The first Nobel Prize
for MS was awarded in 1920. The MS allowed separation of differ-
ent isotopes. More recently, two inventions made it possible to
analyze DNA, peptides and proteins by MS. Matrix-assisted laser
desorption/ionization (MALDI) was invented in 1987. A matrix
a-cyano-4-hydroxycinnamic acid is mixed with an analyte in MAL-
DI-MS. The analyte is desorbed from the matrix with a laser shot
and is ionized [3].
Electrospray ionization technique was invented in 1989 [4]. The
analyte is ionized from a liquid phase into the gas phase in electro-
spray ionization. Thus, liquid chromatography (LC) systems could
be directly interfaced to mass spectrometers. Liquid chromatogra-
phy–tandem mass spectrometry (LC–MS/MS) was applied to pro-
teomics. Peptides were separated in LC and the MS/MS then
records the intact peptides (full MS) before one precursor ion is
selected and fragmented. Fragmentation is commonly produced
 Y.-Y. Zhao, R.-C. Lin / Chemico-Biological Interactions 215 (2014) 7–16 9

(A) UPLC Speed HPLC Speed

(B) UPLC Separation

HPLC Separation

(C) HPLC Intensity

UPLC Intensity

Fig. 2. A typical example was analyzed by UPLC and HPLC method to compared UPLC with HPLC on analytical speed (A), resolution (B) and sensitivity (C). The same six
compounds were separated by UPLC and HPLC methods in 1 min and 4.5 min, respectively (A). For separation, UPLC could separate 4 compounds completely in 1.6 min with a
higher resolution but HPLC could not separate the same compounds in 2.2 min (B). UPLC intensity of compound was 855 with a weak noise but HPLC intensity was 176 with a
strong noise (C).

Fig. 3. Three-dimensional plots of retention time, m/z, and intensity from control white male mouse urine using (A) HPLC–MS with a 2.1 cm  100 mm Waters Symmetry
3.5 lm C18 column, eluted with 0–95% linear gradient of water with 0.1% formic acid/acetonitrile with 0.1% formic acid over 10 min at a flow rate of 0.6 mL/min and (B)
UPLC–MS with 2.1 cm  100 mm Waters ACQUITY 1.7 lm C18 column, eluted with the same solvents at a flow rate of 0.5 mL/min. UPLC has enabled dramatic increases in
chromatographic performance to be obtained for complex mixture separation. This increase in performance was manifested in improved peak separation, increased speed
and high sensitivity. UPLC offers significant advantages over conventional reversed-phase HPLC amounting to a more than doubling of peak capacity, an almost 10-fold
increase in speed and a 3- to 5-fold increase in sensitivity compared to that generated with a conventional 3.5 lm stationary phase.

by argon or nitrogen collision. The fragments are recorded in a MS/
MS spectrum and the fragmentation pattern reveals a specific D
mass for each amino acid in the peptide. With the development
of LC, UPLC, as a novel analytical technology, was produced and ap-
plied to ‘‘omics’’ study. UPLC operates with sub-2 lm chromato-
graphic particles and a fluid system capable of operating at
pressures up to 15,000 psi, providing an increased LC resolution
compared with conventional HPLC using larger particles. The
investigator compared UPLC–MS with conventional HPLC–MS un-
der similar analytical conditions are showed in Fig. 2 and three-
dimensional chromatogram are showed in Fig. 3 [5].
In 2005, Wrona et al. introduced the MSE technique [6], in
which two scanning functions are simultaneously used for collec-
tion data. In the first function, Q1 is scanned from m/z 50–1000,
10 Y.-Y. Zhao, R.-C. Lin / Chemico-Biological Interactions 215 (2014) 7–16

Table 1
UPLC–MSE-based proteomic applications for discovering disease biomarkers in clinical research, animal model and cell model research.

Application Metabolite association Specimen types Analytical methods References

E
Bipolar disorder 36 proteins Cells, human serum UPLC–MS [19]
Major depressive disorder 90 phosphopeptides Human brain tissue UPLC–MSE [20]
Schizophrenia 72 phosphoproteins Human serum IMAC, UPLC–MSE [22]
Psychiatric disorders 488 proteins Human prefrontal cortex GE, UPLC–MSE [23]
tissue
Pituitary disorders 1007 proteins Human pituitary tissue UPLC–MSE [24]
Colorectal cancer 56 proteins Human colorectal tissue UPLC–MSE [25]
Chronic myeloid leukemia 54 responsive and 63 resistant proteins Human bone marrow UPLC–MSE [27]
plasma
Ankylosing spondylitis Monocyte protein Human peripheral blood 2D GE, UPLC–MSE [28]
cells
E
Periodontitis L-plastin, Annexin-1 Human gingival crevicular UPLC–MS [29]
fluid
Spinal cord injury 638 proteins Human seminal plasma 2DSDS–PAGE, UPLC–MSE [30]
Cystic fibrosis 638 different proteins Human temporal bone 2DE, UPLC–MSE [31]
tissue
Inner ear disorders 20 proteins in inner ear and 8 proteins in Human inner ear, otic UPLC–MSE [32]
otic bone bone
E
Type 2 diabetes 1584 proteins Mouse liver ER UPLC–MS [33]
Haloperidol or olanzapine treatment for 1272 proteins Brain UPLC–MSE [34]
schizophrenia
Cisplatin resistance in neuroblastoma 480 proteins Neuroblastoma cell 2DE, MALDI-TOF MS, UPLC– [35,36]
MSE
Respiratory syncytial virus 1352 proteins HEp2 cell 2DSDS–PAGE, UPLC–MSE [37]
CP 93 extracellular proteins Strains 1002 and C231 UPLC–MSE [38]
CP 11 exoproteins Strains 1002 and C231 2DE, MALDI-TOF MS, UPLC– [39]
MSE
Gatifloxacin 12 proteins Yeast 2DE, MALDI-TOF MS, AC, [40]
UPLC–MSE
Botulinum neurotoxin type/G BoNT, NTNH, HA70, HA17 proteins Clostridium argentinense 1D SDS–PAGE, UPLC–MSE [43]
strain

2DE: two-dimensional electrophoresis; 2DGE: two-dimensional gel electrophoresis; 2DSDS–PAGE: two-dimensional sodium dodecyl sulfate–polyacrylamide gel electro-
phoresis; AC: affinity chromatography; CP: Corynebacterium pseudotuberculosis; CRC: colorectal cancer; ER: endoplasmic reticulum; GE: gel electrophoresis; IMAC: immo-
bilised metal ion affinity chromatography.

and Q2 (collision cell) uses a normal low collision energy that pro-
vides for transmission of intact ions through cell collisions. These
ions are then pushed into quadrupole time-of-flight (QTOF) ana-
lyzer and detected with high resolution and mass accuracy. The
second scan function also scans Q1 over the same mass range;
however, Q2 now has a high collision energy that fragments all
of the ions transmitted through Q1. The resulting ions are again de-
tected in the QTOF analyzer. In this way, two mass chromatograms
are generated, one with information on the intact molecules from
the Q1 function, and the other with the fragmented ion informa-
tion from the Q2 function. A variety of data-processing algorithms
can be used to extract metabolite information from these data [7].
The benefits of MSE have been shown to be: MSE can provide par-
allel alternating scans for acquisition at either low collision energy
to obtain precursor ion information (MS) or high collision energy to
obtain full-scan accurate mass fragment, precursor ion and neutral
loss information (MSE), providing similar information to conven-
tional MS2 (MS/MS) in one analytical run. This ability is of major
importance, as it offers the structural information required for
the identification of unknown biomarkers in the context of untar-
geted analyses [8].
Advanced LC–MS improves coverage, sensitivity and through-
put and helps address many key needs for proteomics and
metabolomics. Multiple LC techniques and their continuous
improvements in separation components as well as their hyphened
technologies are providing further advances and enabling increas-
ingly effective large-scale proteomics and metabolomics. However,
as requirements for analytical high throughput and sensitivity in-
crease, the need for even more sensitive and faster LC techniques
continues to discover disease biomarkers. Recently, the UPLC–
MSE technique has been proven to be a powerful and reliable ana-
lytical approach for top-down proteomics study, especially this
method could substantially overcome ion suppression shortcom-
ing for quantitative proteomics and disadvantage of bottom-up
proteomics method [9–12]. UPLC–MSE is becoming increasingly
popular in the analysis of biological fluids in the field of metabolo-
mics because they provide high resolution, accurate mass mea-
surement and structural information [13–18]. UPLC–MSE-based
proteomics and metabolomics applications for discovering bio-
markers of various diseases in clinical research, animal model
and cell model research were summarized in Tables 1 and 2.
5. UPLC–MSE-based proteomics application in disease
biomarker discovery
5.1. UPLC–MSE-based proteomics in clinical research
Clinical proteomics aims to comprehensively identify and
quantify proteins in patient samples to gain insight into the
cellular signaling pathways underlying disease and to discover
novel biomarkers for screening, diagnosis and prognoses to specific
treatments. Table 1 displays UPLC–MSE-based proteomics applica-
tions for discovering biomarkers of various diseases in clinical
chemistry.
Neuropsychiatric diseases including bipolar disorder, major
depressive disorder and schizophrenia are a remarkably complex
disorder with a multitude of behavioral and biological perturba-
tions. Little is known about the molecular mechanisms that are al-
tered in remitting mental illness patients. Bipolar disorder patients
usually experience alternating episodes of hypomania, mania or
depression with symptom-free episodes. Proteome profiling was
investigated for peripheral blood mononuclear cells and serum
from bipolar disorder patients by UPLC–MSE. Cytoskeletal and
 Y.-Y. Zhao, R.-C. Lin / Chemico-Biological Interactions 215 (2014) 7–16 11

Table 2
UPLC–MSE-based metabolomic applications for discovering disease biomarkers in clinical research and animal model research.

Application Metabolite association Specimen Analytical References

types methods
Hepatocarcinoma Glycocholic acid Human urine UPLC–HDMSE [47]
Lung cancer sn-1 lysoPC(16:0), sn-2 lysoPC(16:0), sn-1 lysoPC(18:0), sn-1 lysoPC(18:1), sn-1 Human UPLC–MSE [48]
lysoPC(18:2) plasma
Jaundice syndrome with Alanine, aspartate and glutamate-related metabolites Human urine UPLC–HDMSE [49]
liver disease
Human liver-stagnation and Pentose, glucuronate, ascorbate, aldarate, cysteine, methionine, tyrosine, tryptophan, Human urine UPLC–HDMSE [50]
spleen-deficiency amino sugar, nucleotide sugar
syndrome
Osteoarthritis 284 lipids Human UPLC–HDMSE [51]
plasma
E
Chronic renal failure PC(16:0/18:2), lysoPC(18:1), creatinine, lysoPC(17:0), lysoPC (16:0), Serum UPLC–MS [52,54,58,61]
dihydrosphingosine, tryptophan, ceramide(18:0/16:0), L-acetylcarnitine,
ceramide(18:0/14:0), phytosphingosine
Chronic renal failure Phytosphingosine, adrenosterone, tryptophan, 2,8-dihydroxyadenine, kynurenic acid, Urine UPLC–MSE [52,55,59,62]
creatinine, dihydrosphingosine, dopamine, phenylalanine, ethyl-N2-acetyl-L-
argininate,
N-acetylleucine, 3-O-methyldopa
Chronic renal failure Chenodeoxychrolic acid, palmitic acid, phytosphingosine, lysoPE(18:2/0:0), MG(24:1/ Faece UPLC–MSE [56,60]
0:0/0:0), 12-hydroxy-3-oxocholadienic acid, lysoPE(16:0/0:0), 7-ketolithocholic acid
Chronic renal failure Polyunsaturated fatty acids, indoxyl sulfate, p-cresyl sulfate, allantoin, Kidney tissue UPLC–HDMSE [52,57,63]
phenylacetylglycine, xanthine
Kidney yin deficiency Uric acid, aminoadipic acid, 3-methyldioxyindole, glucosamine, cytidine, indoxyl Urine UPLC–HDMSE [64]
sulfate, cis-aconitic acid, creatinine, estrone, 3-methoxytyrosine, 3-methylguanine
E
Hepatoprotective effect of Glycocholate, 2-Hydoxybutanoic acid Urine UPLC–HDMS [65]
scoparone
Insomnia and treatment of Serotonin, melatonin, prostaglandin D2, 5-hydroxy-L-tryptophan, N-octadecanoyl Drosophila UPLC–HDMSE [66]
Suanzaoren tryptophan
Jujuboside A treatment for 5-Methyl tetrahydrofolate, androstenol, adenosinetriphosphate, serotonin, antepan, Drosophila UPLC–HDMSE [67]
insomnia uridine triphosphate, prostaglandin D2, enkephalin, dopamine, melatonin,
pantothenic acid
MIS and treatment of Wen- Glycolysis or gluconeogenesis, unsaturated fatty acids, fatty acid and purine Urine UPLC–HDMSE [68]
Xin-Formula metabolism
Tianqijiangtang-capsule for Starch and sucrose, pentose and glucuronate interconversions metabolism Urine UPLC–HDMSE [69]
type 2 diabetes
Yeast-induced pyrexia Imidazoleacetate, PE(22:6/20:4), PI(22:5/18:0), indoleacrylic acid, tryptophan, 3- Urine UPLC–HDMSE [70,71]
methyluridine, deoxyadenosine, kynurenic acid, proline, proline betaine, N-
acetylleucine, nicotinamide ribotide, xanthurenic acid
Pinelliae Rhizoma toxicity Phospholipids, amino acids, L-acetylcarnitine, L-carnitine Serum UPLC–MSE [72]
Pinelliae Rhizoma toxicity Phenylacetylglycine, creatinine, deoxycytidine, phenylacetaldehyde, Urine UPLC–MSE [73]
tridecanoylglycine, kynurenic acid, xanthurenic acid, pantothenic acid
Hepatitis C virus Taurine, hypotaurine, ether lipid, glycerophospholipid, arachidonic acid, tryptophan Urine UPLC–HDMSE [74]
Hyperlipidemia Octadecanamide, oleamide, tryptophan, citric acid, ursodeoxycholic acid, creatinine, Urine UPLC–HDMSE [75]
ascorbalamic acid, phenylalanine, 3-O-methyldopa, proline

stress response-related proteins are associated with cell death/sur-
vival pathways in mononuclear cells, while inflammatory response
was related to serum samples. These results suggested that bipolar
disorder patients carry a peripheral fingerprint that has harmful ef-
fects on cell function and that could be used to distinguish bipolar
disorder patients from healthy subjects despite being in a remis-
sion phase. It is hoped that additional study of bipolar disorder pa-
tients in the manic and depressed stages could lead to the
identification of a molecular fingerprint that could predict episodic
switching and for guiding treatment strategy [19]. Major depres-
sive disorder is characterized by feelings of self-esteem and low
mood and by loss of interest in activity. Tissue extracts were ana-
lyzed UPLC–MSE and 5195 phosphopeptides were identified. 90
proteins showed differential phosphorylation in patient tissues.
The majority of these phosphorylated proteins were related to syn-
aptic transmission and cellular architecture not only discovering
potential biomarker candidates but mainly highlighting the major
depressive disorder pathobiology [20]. Schizophrenia is character-
ized by complex and dynamically interacting perturbations in mul-
tiple neurochemical systems. A lot of theories have been proposed
over the years that aim to conceptualize the pathological processes
inherent to schizophrenia, including altered neurotransmission
and signal transduction, autoimmune dysfunction, neuropeptides.
[21]. The report showed both hyper-serotonemia and hypo-sero-
tonemia were related to the longitudinal course of schizophrenia,
suggesting a disturbance of 5-hydroxytryptamine function. How-
ever, evidence of these alterations has been collected piecemeal,
limiting our understanding of the interactions among relevant bio-
logical systems. Therefore, new biomarkers with higher sensitivity
and specificity are waiting to emerge. Jaros et al. used immobilized
metal ion affinity chromatography for enrichment of phosphopro-
teins combined with label-free UPLC–MSE for identification and
measurement of protein and phosphoprotein levels. The analysis
showed 35 proteins from immobilized metal ion affinity chroma-
tography fractions and 72 phosphoproteins from enriched fraction
were altered in schizophrenia patients. This study showed that
schizophrenia patients feature serum abnormalities in phosphory-
lation of proteins involved in acute phase response and coagulation
pathways [22]. In addition, dorsolateral prefrontal cortex proteome
from 12 postmortem brain patients was also analyzed using gel
electrophoresis (GE) and label-free UPLC–MSE. 488 proteins were
identified and involved predominantly in cytoskeletal architecture,
metabolism, transcription/translation and synaptic function [23].
Pituitary proteins were analyzed to provide new insight into pitu-
itary-related disorders mechanism in the above-mentioned re-
search group. Identified 1007 proteins consisted predominantly
12 Y.-Y. Zhao, R.-C. Lin / Chemico-Biological Interactions 215 (2014) 7–16
of enzymes, transporters, transcription/translation factors, cell
structure and secreted proteins [24].
Colorectal cancer (CRC) is one of the most frequently occurring
malignancies in the world, and its prognosis at early stages is poor.
56 proteins from insoluble fractions were differentially expressed
between the tumor and adjacent normal tissue. The connections
between these proteins are involved in reciprocal networks associ-
ated with tumorigenesis, cancer incidence based on genetic disor-
der and skeletal and muscular disorders. Further validation of a
panel of proteins (JUP, KRT5, COL6A1 and TUBB) confirmed the dif-
ferential expression. These proteins gave specific network informa-
tion for CRC, and yielded a panel of novel biomarkers and potential
targets for treatment [25]. Yang et al. compared the patterns of cys-
teine oxidation in the membrane fractions between the tumor and
non-tumor CRC patient tissues. 31 proteins including 37 oxidation-
sensitive cysteines were identified by UPLC–MSE. These proteins
were observed with IAM-binding cysteines in non-tumoral region
more than tumoral region from CRC patients. The protein networks
showed redox status is altered by oxidative stress, perhaps leading
to changes in cellular functionality that promotes tumorigenesis
[26].
Chronic myeloid leukemia is a hematopoietic disorder that is
currently considered incurable. The tyrosine kinase product of
the Philadelphia chromosome (P210 BCR-ABL) provided a pathoge-
netic explanation for the initiation of the chronic myeloid leukemia
chronic phase and was the molecular therapeutic target for the dis-
ease. Protein expression was identified from chronic myeloid leu-
kemia patients using UPLC–MSE proteomic approach. Oxidative
lipid metabolism and regulation of the switch from canonical to
noncanonical WNT signaling may contribute to chronic myeloid
leukemia resistance in the bone marrow compartment [27].
Ankylosing spondylitis (AS) is an inflammatory rheumatic
disease. Clinical hallmarks of ankylosing spondylitis include enthe-
sitis, uveitis, sacroiliitis and persistent spinal inflammation. AS
remains difficult to diagnose and the pathogenic mechanisms of
disease causation and perpetuation are poorly understood. A ser-
um proteomic platform including 2D GE and UPLC–MSE analysis
has been used to distinguish AS patients, rheumatoid arthritis pa-
tients and healthy subjects by peripheral blood monocytes. The
beta subunit of proteasome activator 28 was increased in AS
monocytes. The vascular endothelial growth factor, leucocyte
extravasation, integrin and Toll-like receptor signaling pathways
were associated with rheumatoid arthritis monocytes and AS pa-
tients. Increased endoplasmic reticulum stress response pathway
was not found in either rheumatoid arthritis monocytes or AS pa-
tients. Finally, the proteasome activator 28 complex was shown to
increase the generation of human leucocyte antigen B27 antigenic
epitopes by proteasome. The results demonstrated monocytes play
an important role in the pathogenesis of rheumatoid arthritis and
AS patients [28]. In addition, UPLC–MSE proteomic approach was
applied to periodontal disease, spinal cord injury, cystic fibrosis
and inner ear disorders [29–32].
5.2. LC–MSE-based proteomics in animal model or cell model research
The endoplasmic reticulum (ER) plays a crucial role in the reg-
ulation of the cellular response to insulin. ER stress has been
known to decrease the insulin sensitivity of the liver and lead to
type 2 diabetes. Park et al. performed proteomics of mouse liver
ER by UPLC–MSE. 1584 proteins were identified in control and type
2 diabetic mice livers. Comparison of the rough ER and smooth ER
proteomes from normal mice showed that proteins included pro-
tein synthesis and metabolic processes were enriched in the rough
ER, while those related to transport and cellular homeostasis were
localized to the smooth ER. In addition, proteins included protein
folding and ER stresses were found only in the rough ER. Rough
ER and smooth ER were severely disrupted, including the capacity
to resolve ER stress in mouse livers [33].
Haloperidol and olanzapine are widely used antipsychotic drugs
for schizophrenia and other psychotic disorders. UPLC–MSE proteo-
mics was employed to identify differentially proteins in rat frontal
cortex following subchronic treatment with haloperidol or olanza-
pine. The profiling identified 531 and 741 annotated proteins in
fractions I (cytoplasmic-) and II (membrane enriched-) in two drug
treatments. 59 proteins were altered significantly by haloperidol
treatment, 74 by olanzapine and 21 were common to both treat-
ments. Pathway analysis revealed that both drugs altered similar
classes of proteins associated with cellular assembly/organization,
nervous system development/function and neurological disorders.
The haloperidol results showed a stronger association with Hun-
tington’s disease signaling, while olanzapine showed stronger ef-
fects on glycolysis/gluconeogenesis. This could either relate to a
difference in clinical efficacy or side effect profile of the two drugs
[34].
Neuroblastoma is one of the most aggressive solid tumors in
childhood. Therapy resistance to anticancer drugs represents the
major limitation to the effectiveness of clinical treatment. Human
neuroblastoma cell SH-SY5Y and its cisplatin resistant counterpart
were studied by 2-DE electrophoresis-MS and label-free UPLC–MSE
proteomic approach. The results demonstrated nuclear factor-ery-
throid 2-related factor 2 (Nrf2) pathway play a protective role in
normal cells and may represent a potential novel target to control
cisplatin resistance in neuroblastoma [35]. Confocal microscopy
experiments, enzyme assay, and Western blotting of proteins reg-
ulated by Nrf2 provided evidences that Nrf2 pathway in cisplatin-
resistant neuroblastoma cell line. Western blotting showed the
increment of Nrf2 in a resistant cell line was clearly evident. Con-
focal microscopy experiment showed the Nrf2 signal was mostly
distributed in the cytoplasm both in the sensitive cell line and in
the resistant cell line. This result was in agreement with Nrf2 acti-
vation and translocation into nuclei in the resistant cell line. In
addition, proteins that are known to be under the control of Nrf2,
antioxidant genes peroxiredoxins were found modulated by cur-
rent proteomic analysis, and in the case of peroxiredoxin 1, the
expression change level was validated by Western blotting [35].
Other evidence of the activation of Nrf2 down stream enzymes
came from the measurements of glutathione peroxidase 1 activity.
Results revealed an increased activity in resistant cell line com-
pared to the sensitive cell line. Another UPLC–MSE proteomics
was applied to study the cellular response to curcumin in a
SH-SY5Y cell line sensitive to cisplatin [36]. The results showed
that sixty-six proteins were different expressed in response to cur-
cumin treatment in sensitive cells, whereas thirty-two proteins
were significantly modulated in treated resistant cells. Functional
analysis demonstrated that proteins involved in cellular assembly
and organization, biosynthesis and glycolysis were down-regu-
lated by curcumin treatment. Proteome changes were associated
to cell cycle arrest in the G2/M phase and accumulation of poly-
ubiquitinated proteins.
Human respiratory syncytial virus is a pathogen of the family of
Paramyxoviridae, causing severe infection of the lower respiratory
tract predominantly in young children and the elderly. A quantita-
tive study was carried out to compare the proteome of respiratory
syncytial virus infected versus uninfected cells to determine new
pathways regulated during viral infection. The purified peptides
were characterized by UPLC–MSE. 1352 proteins were identified
and their abundance compared between infected and non-infected
cells. Synthesis of interferon-induced protein with tetratricopep-
tide repeats 3 (IFIT3) and 50 -30 -exoribonuclease 2 (XRN2) mRNAs
were found to be highly induced upon respiratory syncytial virus
infection in a time dependent manner. Accordingly, IFIT3 protein
accumulated during the time course of infection. In contrast, little
 Y.-Y. Zhao, R.-C. Lin / Chemico-Biological Interactions 215 (2014) 7–16
variation was observed in XRN2 protein, but different forms were
present in infected versus non-infected cells [37].
Bacterial exported proteins represent key components of the
host-pathogen interplay. UPLC–MSE was employed for protein
identification and quantification form Corynebacterium pseudotu-
berculosis (CP). 93 proteins were identified with high confidence
and 44 proteins were commonly identified in two different strains.
Comparative analyses of the exoproteomes of two CP strains were
helpful to gain novel insights into the contribution of the exported
proteins in the virulence of this bacterium [38]. This research
group identified 45 extracellular proteins. The comparative
analysis between the strains 1002 and C231 identified 13 and 3
strain-specific proteins, respectively, 11 of which are novel [39].
In addition, UPLC–MSE proteomic approaches were applied to
study gatifloxacin, biotherapeutic proteins, influenza vaccination,
botulinum neurotoxin type/G, bacterial endosymbiont, Marek’s
disease virus and cysteine oxidation [40–46].
6. Metabolomics application in disease biomarker discovery
6.1. UPLC–MSE-based metabolomics in clinical research
Cancer biomarker discovery is important for early diagnosis,
disease mechanism elucidation, and targeted therapy for the dis-
ease. Ultra-performance liquid-chromatography coupled with
high-definition MSE (UPLC–HDMSE) metabolomics was used to
identify and measure the metabolite profile of glycocholic acid
from hepatocarcinoma patients. Urinary glycocholic acid expres-
sion was increased and primary and secondary bile acid biosynthe-
sis and bile secretion were disturbed [47]. LysoPCs can be a clinical
diagnostic indicator that uncovered pathophysiological changes.
Dong et al. have discriminated between different types of lysoPCs
from lung cancer patients and healthy subjects. 14 pairs of plasma
lysoPCs regioisomers were identified. All lung cancer patients had
the same 5 lysoPCs metabolic abnormalities, specifically in sn-1
lysoPC(18:1), sn-1 lysoPC(18:2), sn-1 lysoPC(18:0), sn-1 ly-
soPC(16:0) and sn-2 lysoPC(16:0). Thus, the function of isomers
may be related to lung cancer [48].
Understanding syndromes is a core study to develop more effi-
cient therapeutic strategies, classification, and diagnostic criteria
for patients. Pharmacological study and clinical practice have
shown that patients with liver disease are complicated by jaundice
syndrome. Wang’s study established UPLC–HDMSE metabolomics
for study the metabolic profiling of jaundice syndrome patients
with liver disease. 44 metabolites were identified from jaun-
dice syndrome. Aspartate, glutamate and alanine metabolism and
synthesis and ketone body degradation were found to be perturbed
in jaundice syndrome patients [49].
Metabolite profiling of human spleen-deficiency and liver-stag-
nation syndrome was performed by UPLC–HDMSE. 12 urinary
metabolites were identified involving metabolic pathways of
pentose and glucuronate interconversions, aldarate, ascorbate,
tryptophan, methionine, cysteine, tyrosine, nucleotide sugar and
amino sugar metabolism. L-homocystine, prolylhydroxyproline,
a-N-phenylacetyl-L-glutamine and 2-octenoylcarnitine were effec-
tive for the diagnosis of human spleen-deficiency and liver-stagna-
tion syndrome, with an 93.0% sensitivity [50]. Other investigators
developed an UPLC–MSE approach to analyze lipid profiling from
osteoarthritis patients. Lipid metabolism associated with osteoar-
thritis and the release of arachidonic acid from phospholipids [51].
6.2. UPLC–MSE-based metabolomics in animal model research
UPLC–based metabolomics has been used to study kidney dis-
eases in the last several years [52]. A series of experimental studies
13
have been performed on chronic kidney disease (CKD) animal
models to investigate the metabolic profiling of serum, urine, feces
and kidney tissues, and these results have led to new insights into
the development of CKD [52]. The adenine-induced CKD model has
the advantage of being more similar to the development of human
chronic renal injury in comparison to genetic models, and these
models mirror the progression of renal injury after a prolonged
period of development [53]. Increased serum lysoPC (18:1),
PC(16:0/18:2), creatinine, lysoPC (16:0) and lysoPC(17:0) and de-
creased serum tryptophan, dihydrosphingosine, ceramides (18:0/
16:0), L-acetylcarnitine, ceramides (18:0/14:0) and phytosphingo-
sine were observed in adenine-induced CRF rats. Furthermore,
CRF could be predicted according to various metabolites including
lysoPC(18:1), lysoPC(17:0), ceramides (18:0/16:0), lysoPC (16:0),
creatinine, ceramides (18:0/14:0) and tryptophan [54]. The urine
of CRF rats were characterized by increased adrenosterone, phyto-
sphingosine, tryptophan, creatinine, 2,8-dihydroxyadenine and
dihydrosphingosine, together with decreased 3-O-methyldopa,
N-acetylleucine, dopamine, ethyl-N2-acetyl-L-argininate, kynure-
nic acid and phenylalanine [55]. The altered metabolites demon-
strated perturbations of phospholipids, amino acids and
creatinine metabolism in the CRF rats. These results provided evi-
dence for the complex perturbation of phospholipids, amino acids
and creatinine metabolism in CKD. It was reported that changes in
the fecal metabolite profile, such as palmitic acid, MG(24:1/0:0/
0:0), chenodeoxychrolic acid, 12-hydroxy-3-oxocholadienic acid,
phytosphingosine, lysoPE(16:0/0:0), lysoPE(18:2/0:0) and 7-keto-
lithocholic acid, could be used as early biomarkers for adenine-in-
duced CRF rats [56]. Kidney metabolomics based on the
UPLC–HDMSE was calculated to explore the excretion pattern in
the adenine-induced CRF rats. The results showed that the most
important CRF-related metabolites were polyunsaturated fatty
acids, p-cresyl sulfate and indoxyl sulfate. p-cresyl sulfate and in-
doxyl sulfate were significantly increased in CRF rats [57]. Further,
the above-mentioned UPLC-based metabolomics method was ap-
plied to therapeutic effect of ergone. The results showed that some
biomarkers were completely reversed by ergone [58–60]. In addi-
tion, the results also showed that several biomarkers were re-
versed completely by the surface layer of Poria cocos [61–63].
UPLC–HDMSE metabonomics was also undertaken to explore thy-
roxine and reserpine-induced kidney yin deficiency and the thera-
peutic effect of Liu Wei Di Huang Wan [64].
Natural medicines, widely used for the treatment of a variety of
diseases, have recently attracted the interest of the modern scien-
tific community as alternative therapy. Scoparone is an important
constituent of Artemisia annua L., and displayed bright prospects in
the prevention and therapy of liver injury. The effects and possible
mechanisms of scoparone against CCl4-induced liver injury were
studied by UPLC–HDMSE metabolomic approach. The identified
metabolites were associated with pyrimidine metabolism and pri-
mary bile acid biosynthesis. Scoparone has a potential pharmaco-
logical effect through regulating multiple disturbed pathways to
the normal level [65]. Suanzaoren decoction was used to treat
insomnia, and its mechanism remains unclear. UPLC–HDMSE
metabonomics was study to explore globally metabolomic charac-
ters of the insomnia and the therapeutic effects of suanzaoren
decoction. The identified metabolites were associated with pertur-
bations in amino acid and fatty acid metabolism, in response to
insomnia through immune and nervous system. The result showed
suanzaoren decoction increases sleep activity and exhibits binding
affinity for serotonin receptors. The therapeutic effects of suanzao-
ren decoction may mediate through serotonergic activation [66].
Other study showed metabolic profiling was restored to their
baseline values after Jujuboside A treatment for insomnia [67].
UPLC–MSE was designed to explore metabolomic characters of
the myocardial ischemia syndrome (MIS) and the therapeutic
14 Y.-Y. Zhao, R.-C. Lin / Chemico-Biological Interactions 215 (2014) 7–16
effects of Wen-Xin-Formula. 17 biomarkers were identified and
metabolic pathway analysis suggested that the biosynthesis of
unsaturated fatty acids metabolism, glycolysis or gluconeogenesis
metabolism, purine metabolism and fatty acid biosynthesis
networks were acutely disturbed by MIS. Wen-Xin-Formula has
potential pharmacological effect through regulating multiple met-
abolic pathways to normal state [68]. The efficacy and mechanism
of Tianqijiangtang-capsule for type 2 diabetes was evaluated by
UPLC–MSE metabolomic approach. The changes metabolites sug-
gested that the disorders of pentose and glucuronate interconver-
sions and starch and sucrose metabolism are related to type 2
diabetes and the potential effect of Tianqijiangtang-capsule on all
of these metabolic pathways. This work will provide better under-
standing of the mechanism of Tianqijiangtang-capsule in clinical
use [69]. Fever is a prominent feature of many diseases. Fever
can be easily judged by body temperature in clinic; however, the
pathogenesis of fever is still not well clear. A febrile response is a
systemic pathological process that can cause metabolic disorders.
UPLC–MSE metabolomics was employed to investigate the urinary
biochemical characteristics of yeast-induced pyrexia rats. 16
metabolites were identified as potential biomarkers. The thermo-
regulatory circuitry of ‘‘endogenous pyrogen"-hypothalamus Na+/
Ca2+-cAMP"’’ was confirmed. The disturbance of tryptophan
metabolism might be one of the important mechanisms in explain-
ing the biochemical basis of the febrile response [70]. Further, this
method was applied to therapeutic effect of Qingkailing injection.
The results demonstrated that the antipyretic effect of Qingkailing
injection was performed by repairing the perturbation of amino
acids metabolism [71].
The toxicity of natural medicines was also evaluated by UPLC–
MSE metabonomic approach. Pinelliae Rhizoma (PR) is used to treat
cough, vomiting, infection and inflammation. The toxicity of PR in
rats was evaluated by UPLC–MSE. The serum amino acids, phos-
pholipids, L-acetylcarnitine and L-carnitine showed significant
differences after oral administration, which indicated the perturba-
tions of phospholipid metabolism, amino acid metabolism and car-
nitine metabolism in PR-induced rats [72]. Further, urinary
metabonomics was used to elucidate metabolome of rats induced
by PR. 10 identified biomarkers indicated the perturbations of
phenylacetylglycine tryptophan and pantothenic acid metabolism
in PR-induced rats [73]. In addition, hepatitis C virus and hyperlip-
idemia rats were studied by UPLC–HDMSE metabolomics [74,75].
7. Conclusion and perspectives
The development and application of proteomics and metabolo-
mics has increased tremendously over the decade. LC techniques
coupled with MS improve coverage, sensitivity, and throughput
and help address many key needs for proteomics and metabolo-
mics research. the disease biomarker discovery need to analyze
vast samples to obtain statistically relevant results demands
high-throughput analytical platforms where LC separations must
achieve maximal peak capacity in a short time period. With the
emergence of more effective UPLC–MSE technologies and the vari-
ety of fractionation approaches, the proteomics and metabolomics
have been greatly expanded. It offers significant potential for the
discovery of novel candidate biomarkers from disease samples.
Currently there is no single platform that represents the perfect
technology for proteomics and metabolomics applications, and
integration of multiple technologies is often required for detection
and quantitation of low abundance proteins and metabolites. The
improvement of dynamic range, high throughput, reproducibility
and quantitation will further demand to promote technology
development and improvement efforts. The new separation and
analysis technologies with sensitivity and accuracy including UPLC
separations, ultra performance convergence chromatography
(UPC2), and high efficiency QTOF/MS/MSE presently appear promis-
ing for future discovery platforms and applications. With improve-
ments in quantitation accuracy, throughput, and robustness, the
UPLC–MSE-based proteomics and metabolomics platform may
eventually become a powerful tool for biomarkers discovery, dis-
ease diagnosis and targeted therapeutics that provides simulta-
neous measurements of many disease relevant analytes.
The informatics and statistical analysis is an important part of
proteomics and metabolomics platform. The effective software will
be necessary for processing enormous datasets, which may involve
run-to-run feature alignment, peak detection, intensity normaliza-
tion, feature matching to the database, and statistical analysis to
generate a list of high confidence potential biomarkers.
Based on the complexity and challenge of proteomics and met-
abolomics study, especially in large scale clinical application, coop-
eration from different laboratories may be required for standard
and better validation of the discovery results and reducing poten-
tial biases. The common standards are needed so that platform per-
formance in different laboratories may be easily compared and
proteomics results can be effectively shared and used. For proteo-
mics and metabolomics, further developments of LC would likely
have significant benefits for broad areas of application. Gene,
protein and metabolite interact to execute molecular processes in
biological systems. These complex interactions must not be exclu-
sively addressed by a simplified method. The combined use of
proteomics and metabolomics permits a more holistic view of bio-
logical systems and their alterations in disease.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This study was supported by Program for New Century
Excellent Talents in University, China (No. NCET-13-0954) and
Changjiang Scholars and Innovative Research Team in University,
China (No. IRT1174) National Natural Science Foundation of China,
China (Nos. J1210063, 81001622, 81073029), As a Major New Drug
to Create a Major National Science and Technology Special, China
(Nos. 2011ZX09401-308-034, 2014ZX09304-307-02), China
Postdoctoral Science Foundation, China (No. 2012M521831), Key
Program for the International S&T Cooperation Projects of Shaanxi
Province, China (No. 2013KW31-01), Natural Science Foundation of
Shaanxi Provincial Education Department, China (No. 2013JK0811)
and Administration of Traditional Chinese Medicine of Shaanxi,
China (No. 13-ZY006).
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