Identification of urinary biomarkers for type 2 diabetes usingbead-based proteomic approach

Lina Chu a, Guangzhen Fu a, Qian Meng a,b, Hui Zhou a,b, Man Zhang a,b,*aThe Ninth Clinical Medical College of Peking University, Beijing Shijitan Hospital, Beijing, ChinabDepartment of Clinical Laboratory, Beijing Shijitan Hospital, Capital Medical University, Beijing, China

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 1 ( 2 0 1 3 ) 1 8 7 – 1 9 3

a r t i c l e i n f o

Article history:

Received 19 February 2013

Received in revised form

23 April 2013

Accepted 17 May 2013

Available online 13 June 2013

Keywords:

Type 2 diabetes mellitus

Urinary peptides

Biomarkers

MALDI-TOF MS

a b s t r a c t

Aims: To seek urinary peptides as biomarkers distinguishing type 2 diabetes mellitus

(T2DM) patients from healthy controls.

Methods: Random urine samples obtained from 28 patients with T2DM and 29 healthy

individuals were analyzed by matrix-assisted laser desorption ionization time-of-flight

mass spectrometry (MALDI-TOF MS) after purification using weak cationic-exchange mag-

netic beads (MB-WCX). Then the generated mass spectra of peptides were analyzed by

ClinProTools2.1 bioinformatics software. Subsequently, the amino acid sequences of differ-

ently expressed peptides were identified by a nano-liquid chromatography–tandem mass

spectrometry and a Sequest search found the corresponding protein name.

Results: Three differently expressed peptides and their mass to charge ratios (m/z) were

found. Compared with healthy controls, the peak areas of the three differently expressed

peptides were all reduced in T2DM, and the m/z were 1056.1 (m/z), 1963.5 (m/z), 2123.5 (m/z),

respectively. The above-mentioned peptides were further identified as fragments of histi-

dine triad nucleotide-binding protein 1 (HINT1), bifunctional aminoacyl-tRNA synthetase

(EPRS), and clusterin precursor protein (CLU).

Conclusions: Histidine triad nucleotide-binding protein 1, bifunctional aminoacyl-tRNA syn-

thetase, and clusterin precursor protein may serve as potential biomarkers distinguishing

type 2 diabetes mellitus patients from healthy controls.

# 2013 Elsevier Ireland Ltd. All rights reserved.

Contents available at Sciverse ScienceDirect

Diabetes Researchand Clinical Practice

journal homepage: www.elsevier.com/locate/diabres

1. Introduction

Type 2 diabetes mellitus (T2DM) is a serious and growing public

health problem, which is characterized by hyperglycemia,

* Corresponding author. Tel.: +86 010 63926389; fax: +86 1063926283.E-mail address: mzhang99@aliyun.com (M. Zhang).

Abbreviations: HbA1C, glycated hemoglobin; MALDI-TOF MS, matrtrometry; MB-WCX, weak cationic-exchange magnetic beads; m/z, mas1; EPRS, bifunctional aminoacyl-tRNA synthetase; CLU, clusterin precuglucose; ROS, reactive oxygen species; MSC, multi-tRNA synthetaseNSAP1, NS1-associated protein 1; GAIT, IFN-g activated inhibitor of trafactor A; DAPK, death associated protein kinase.0168-8227/$ – see front matter # 2013 Elsevier Ireland Ltd. All rights

http://dx.doi.org/10.1016/j.diabres.2013.05.004

absolute or relative lack of insulin. It involves systemic glucose,

lipid, and protein metabolic disorders. In order to monitor and

treat complications promptly, regular re-examinations are

essential to T2DM patients [1,2]. In routine clinical practice,

ix-assisted laser desorption ionization time-of-flight mass spec-s to charge ratio; HINT1, histidine triad nucleotide-binding proteinrsor protein; T2DM, type 2 diabetes mellitus; FPG, fasting plasma

complex; GAPDH, glyceraldehydes-3-phosphate dehydrogenase;nslation; Cp, ceruloplasmin; VEGF-A, vascular endothelial growth

reserved.

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 1 ( 2 0 1 3 ) 1 8 7 – 1 9 3188

blood glucose monitoring is frequently used to evaluate the

efficacy of drugs and facilitate drug dose adjustment. However

blood glucose concentrations are influenced by diurnal varia-

tion, venous stasis, posture, prolonged fasting, and acute stress

[3,4].

Urinary proteomics is emerging as a powerful diagnostic

and prognostic tool in both kidney and systemic disease [5,6].

The human urine contains more than 1500 different proteins

and also many more peptide fragments. Considering the

generation of small peptide fragments in urine may be the

effect of proteolytic degradation, analysis of small peptides

might be better suited as an approach to diagnose systemic

disease compared with large molecular weight proteins [7,8].

Further these small molecular weight proteins can be

analyzed in a mass spectrometer without additional manipu-

lation (e.g. tryptic digestion) [9].

CLINPROTTM MALDI-TOF MS applies different functional

magnetic beads to process various complex body fluids and

then employs MALDI-TOF MS to discover potential biomarkers

(native small peptides) associated with diseases [10,11].

In this study, we used weak cationic-exchange beads and

MALDI-TOF MS to access the urine peptidome profiles of T2DM

patients and healthy controls, with the aim of identifying

potential urinary peptides distinguishing T2DM patients from

healthy controls.

2. Subjects, materials and methods

2.1. Study subjects

Patients with T2DM and healthy controls were recruited from

Beijing Shijitan Hospital from February 2012 to June 2012.

The participants all gave informed consent, in accordance

with the provisions of the Helsinki Declaration and approved

by the ethics committee of Shijitan Hospital. Finally twenty-

eight patients with T2DM and twenty-nine healthy controls

meeting inclusion and exclusion criteria were selected into

our study. The T2DM group had a fasting plasma glucose (FPG)

�7.0 mmol/l and glycated hemoglobin (HbA1C) �6.0%

(42 mmol/mol) and no complications. In the control group,

FPG was between 3.9 and 6.1 mmol/l. The urine samples of all

subjects had no hematuria, ketosis, and urinary albumin/

creatinine ratio (A/Cr) was less than 30 mg/g.

The clinical characteristics of selected subjects are shown

in Table 1. Among the two groups of people, gender, age,

Table 1 – Clinical characteristics of T2DM and healthycontrols.

T2DM Healthy controls p

Gender (M/F) 21/7 23/6 0.698

Duration (year) 5.05 � 4.25 – –

Mean age � SD 53.50 � 9.72 50.93 � 9.94 0.193

FPG (mmol/l) 9.21 � 1.87 5.49 � 0.35 0.000

CHOL (mmol/l) 5.45 � 1.14 5.04 � 0.78 0.116

TRIG (mmol/l) 1.48 � 0.59 1.20 � 0.48 0.064

HbA1C (% mmol/l) 7.7 � 1.4

61 � 15

5.2 � 0.3

33 � 3

0.000

A/Cr (mg/g) <30 mg/g <30 mg/g –

cholesterol (CHOL), triglyceride (TRIG) and A/Cr did not differ

significantly.

2.2. Urine samples preparation

The random urine samples were collected from volunteers in

the morning, kept at 4 8C and were transferred to the

laboratory within 2 h, centrifuged at 400 � g for 5 min, and

the supernatant collected, divided in aliquots and frozen at

�80 8C refrigerator until use. Prior to urine peptides isolation,

urine samples were thawed at 4 8C followed by centrifugation

at 3000 rpm for 10 min, and then supernatants immediately

proceeded to peptides fractionation.

2.3. Fractionation of urine peptides

Urine samples were subjected to fractionation by using weak

cationic-exchange magnetic beads according to the manu-

factures’ instructions (Bruker Daltonics). Samples were puri-

fied and isolated through three steps: binding, washing, and

elution. 10 ml MB-WCX and 10 ml MB-WCX binding solution

were added in a polypropylene tube and mixed thoroughly.

The tube was placed in a magnetic bead separator (Bruker

Daltonics) to separate the unbound solution. The magnetic

beads were then washed three times. After that another 5 ml

MB-WCX eluting solution was added and mixed intensively.

Finally, the clear supernatant was transferred into a fresh

tube, and 5 ml MB-WCX stabilizing solution was added, the

well mixed eluate was then stored at �20 8C.

2.4. MALDI-TOF MS data acquisition

A portion of eluate was diluted 1:10 in matrix solution

containing a-cyano-4-hydroxycinnamic acid (0.3 g/l in etha-

nol:acetone 2:1, Bruker Daltonics). Then 1 ml of the resulting

mixture was spotted onto the AnchorChip target (Bruker

Daltonics) and allowed to air dry. Ionization was achieved by

irradiation with a nitrogen laser (l = 337 nm) operating at

25 Hz. Before MS analysis, mass calibration was performed.

For each MALDI spot, 400 spectra were acquired in analysis (50

laser shot at 8 different spot positions) and an average of 8

spots represented one urine sample.

2.5. Data processing

The spectra of all signals with a signal-to-noise ratio >5 were

collected in the mass of 1000–10000 Da. All the spectra were

analyzed using ClinProTools2.1 software to normalize spectra

(using total ion count), subtract baseline and determine peak

m/z values and intensities. To align the spectra, a mass shift of

no more than 0.1% was determined. The peak area was used as

quantitative standardization. t-Test was used to analyze

normally distributed continuous data and Wilcoxon test for

non-normally distributed continuous data. In all cases p < 0.05

was accepted as statistically significant.

2.6. Peptide sequence

The sequences of differential expression peptides were

identified using a nano-liquid chromatography–tandem mass

Fig. 1 – Spectrum profiles of all samples. A represents the complete spectra of the type 2 diabetes mellitus patients (red) and

the healthy controls (green). The figure also shows the average urine spectrum profiles of T2DM (red, B) and the healthy

controls (green, C). x-axis: mass to charge ratio (m/z); y-axis: relative intensity; z-axis: samples.

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 1 ( 2 0 1 3 ) 1 8 7 – 1 9 3 189

spectrometry, which consisted of an Aquity UPLC system

(Waters) and a LTQ Obitrap XL mass spectrometer (Thermo

Fisher) equipped with a nano-ESI source. Firstly the selected

peptides were further desalted by use of C18 trap column

(symmetry1 180 mm � 20 mm � 5 mm, nano ACQUITYTM) with

a flow rate of 15 ml/min in 5% acetonitrile (Sigma–Aldrich),

0.05% trifluoroacetic acid (Sigma–Aldrich) for 3 min. Then the

desalted peptides were analyzed by C18 analytical column

(symmetry1 75 mm � 150 mm � 3.5 mm, nano ACQUITYTM) at a

flow rate of 400 nl/min. The mobile phases A (5% acetonitrile,

0.1% formic acid, Sigma–Aldrich) and B (95% acetonitrile, 0.1%

formic acid) were used for analytical columns. Gradient

elution was achieved using 5% B–50% B–80% B–80% B–50%

B–5% B in 100 min. The MS instrument was operated in a data-

dependent model. The range of full scan was 400–2000 m/z

with a mass resolution of 100,000 (m/z 400). The eight most

intense monoisotope ions were the precursors for collision

induced to two consecutive scans per precursor ion followed

by 60 s of dynamic exclusion.

2.7. Bioinformatics and identification of urine biomarkers

The obtained spectra were analyzed with Bioworks Browser

3.3.1 SP1 (Thermo Fisher) and the resulting mass lists were

matched against the IPI Human database (v3.45) using Sequest

search. Parameters were set as follows: Delton � 0.1; charge

2+, Xcorr > 2.6; charge 3+, Xcorr > 3.1; charge >3+, Xcorr > 3.5;

Table 2.1 – Statistical information of different peaks between

Index M/C ratio PTTA(t) P-WTest PAD_1

1 1056.1 0.108 0.049 0.001

36 1963.5 0.054 0.018 0.002

44 2123.5 0.037 0.05 0.5

Note: The p-value of Anderson–Darling test (PAD) can give information a

normally distributed; the p-value of the t-test (PTTA, preferable for nor

preferable for abnormal distributed data) was used to confirm significant

Group1: T2DM; Group2: healthy controls. Ave means average area of pea

parent ion masses tolerance: 10 ppm; fragment ion masses

tolerance: 1 Da.

3. Results

3.1. Unique urine peptidome of T2DM patients andhealthy controls

Urine samples from 28 patients with T2DM and 29 healthy

controls were analyzed by MALDI-TOF MS in order to discover

the urinary peptides biomarkers of interest for T2DM.

A total of 115 peaks were detected by ClinProTools2.1

software in the mass ranging from 1000 to 10,000 Da. The

spectrum profiles are shown in Fig. 1.

3.2. Different urinary peptides between T2DM patientsand healthy controls

In 115 peaks, three differential peaks were found with

statistical significance of p < 0.05 and the m/z were 1056.1,

1963.5, and 2123.5, respectively. The corresponding p-values

were 0.049, 0.018, and 0.037 (Table 2.1). The three differently

expressed peaks were all lower in the T2DM group compared

with the healthy controls. The results are shown in Fig. 2.

As patients with diabetes and healthy controls included

both male and female, we evaluated the three differently

T2DM and healthy controls.

PAD_2 Ave_1 Ave_2 SD_1 SD_2

0.052 96.8 142.4 103.3 107.6

0.081 159.7 203.9 83.8 85.4

0.484 275.9 333 62.4 127.8

bout the normal distribution: <0.05 not normally distributed, >0.05

mal distributed data) or the p-value of the Wilcoxon test (P-WTest,

different. p < 0.05 was accepted as statistically significant difference.

k, SD means standard deviation.

Fig. 2 – Differently expressed urinary peptides in T2DM patients and healthy controls. A is the full sites including three

differential urinary peptide peaks, x-axis: index; y-axis: peak area. The peak area distributions of each differential peak

(1056.1, 1963.5, and 2123.5, respectively) in all samples were shown in B. The average peak area of the three differently

expressed urinary peptides in figure C. Red means T2DM, green means healthy controls.

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 1 ( 2 0 1 3 ) 1 8 7 – 1 9 3190

Table 2.2 – Statistical information of the three different peaks between female and male.

Index M/C ratio PTTA(t) P-WTest PAD_1 PAD_2 Ave_1 Ave_2 SD_1 SD_2

1 1056.1 0.848 0.711 0.007 0.001 147.7 139.4 143.3 135.6

36 1963.5 0.632 0.676 0.006 0.004 257.7 236.8 161 130

44 2123.5 0.161 0.209 0.097 0.018 319.5 404.2 114.4 204.8

Note: The p-value of Anderson–Darling test (PAD) can give information about the normal distribution: <0.05 not normally distributed, >0.05

normally distributed; the p-value of the t-test (PTTA, preferable for normal distributed data) or the p-value of the Wilcoxon test (P-WTest,

preferable for abnormal distributed data) was used to confirm significant different. p < 0.05 was accepted as statistically significant difference.

Group1: female; Group2: male. Ave means average area of peak, SD means standard deviation.

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 1 ( 2 0 1 3 ) 1 8 7 – 1 9 3 191

expressed peaks by sexual grouping. There were no gender

differences with p-values of 0.848, 0.632, and 0.209 respectively

(Table 2.2).

3.3. Identification of amino acid sequence of differentialpeptides

With a nano-liquid chromatography–tandem mass spectrom-

etry detection, the peptide sequence of the differential peaks

were identified and the Sequest search reported the protein

name. The peptides of m/z 1056.1, 1963.5, and 2123.5 were a

partial sequence of histidine triad nucleotide-binding protein

1, bifunctional aminoacyl-tRNA synthetase, and clusterin

precursor, respectively. The complete identification results

are shown in Table 3.

4. Discussion

Urine produced by the kidney allows the human body to

eliminate waste products from blood. The kidney also

maintains whole body homeostasis and produces hormones

including renin and erythropoietin. In 24 h, about 900 l of

plasma flows through the kidneys of which 150–180 l is

filtered. Peptides and small proteins (<10 kDa) are freely

filtered by the glomerulus. However, more than 99% of this

primitive urine is reabsorbed [6,12]. Therefore urine may

contain information not only about the kidney and the urinary

tract but also about more distant organs via plasma obtained

by glomerular filtration. Therefore the study of low molecular

mass proteins and peptides helps to provide new resources to

look for undiscovered biomarkers [13].

Compared with other body fluids, urine can be collected

easily in large quantities using non-invasive procedures. This

allows repeated sampling of the same individual and easy

assessment of reproducibility [7,14]. In addition, the urinary

protein content is relatively stable because proteolytic

degradation by endogenous proteases may be substantially

complete during stagnation for hours in the bladder [12]. This

is significantly different from blood, in which the activation of

Table 3 – Identified peptides sequence of three differential pe

Index m/z Molecular weight Amino sequenc

1 1056.1 1056.108006156 K.KHISQISVAEDDDESLLG

36 1963.5 1963.939597146 K.FAGGDYTTTIEAFISASG

44 2123.5 2121.198092726 F.SLPHRRPHFFFPKSRIV.R

proteases is inevitably associated with its collection and

various products from coagulation cascade will be present in

serum, and these often interfere with MS analysis. Saliva and

tears are collected in low volume and do not reflect the level of

certain plasma proteins. Many proteases and non specific

materials such as food residues or microorganisms exist in

saliva [15]. Urine is a preferred source to discovery biomarkers

of system disease and poses minimal infectious disease risk to

participants and researchers.

Proteomic analyses based on mass spectrometry technique

can discover candidate biomarkers by broadly surveying large

numbers of species in an untargeted pattern. For a variety of

reasons, it is still not possible to analyze the proteome of a

complex biological sample by MS without preceding separa-

tion. Hence, pre-analysis separation is a pre-requisite to

acquire proteins and peptides of interest [16]. In this study, we

chose weak cationic-exchange magnetic beads to purify and

gather acidic proteins and peptides, which are native in urine

without trypsinization. MALDI-TOF MS tends to detect low

molecular mass proteins and peptides (1000–20,000 Da).

T2DM is a multifactorial disease regulated by genetic and

environmental factors. However, there are still many

unknowns about its pathological mechanism. There is

increasing evidence that oxidative stress and inflammation

play critical roles in the pathogenesis of T2DM and the

development of complications. High intra- and extra-cellular

glucose levels result in overproduction of reactive oxygen

species (ROS) in diabetes. The elevated ROS production and

concomitant decline of antioxidant defense mechanisms lead

to endothelial cell injury, increased microvascular permeabil-

ity and release of inflammatory mediators, which further

induce an inflammatory reaction and potentiate a positive

feedback loop [17–20]. Additionally, pancreatic cells are

particularly sensitive to ROS because of the low activities of

enzymes in free-radical quenching [21].

In a comparative analysis of urine peptidome, we found

three down-regulated peptides in the urine of T2DM patients –

histidine triad nucleotide-binding protein1 (HINT1), bifunc-

tional aminoacyl-tRNA synthetase (EPRS), and clusterin

precursor protein (CLU).

aks.

es Protein name

HLMIVGK.K Histidine triad nucleotide-binding protein 1 (HINT1)

R.A Bifunctional aminoacyl-tRNA synthetase (EPRS)

Clusterin precursor (CLU)

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 1 ( 2 0 1 3 ) 1 8 7 – 1 9 3192

HINT1 is ubiquitously expressed in eukaryocytes and

bacteria with an evolutionary highly conserved sequence.

The basic function is to hydrolyze adenosine 50-monopho-

sphoramidate substrates such as AMP-morpholidate. Accu-

mulated evidence shows the HINT1 protein plays an inhibitory

role in a number of gene transcription control pathways, for

example serving as a tumor suppressor participating in

several apoptotic pathways [22–25]. However, the precise

mechanism of action of HINT1 with respect to tumor

suppression is not known, and the potential link between

HINT1 and diabetes has not been reported.

EPRS is responsible for charging the amino acids Glu and

Pro to the 30-end of their cognate tRNAs for initiation of

protein translation, which is the canonical aminoacylation

function. There exist cytosolic and mitochondrial EPRS in

mammalian cells. Many reports demonstrate cytosolic ERPS

also possesses noncanonical function such as specific

translational silencing of inflammatory gene expression.

When stimulated with IFN-g, EPRS is phosphorylated and

released from the multi-tRNA synthetase complex (MSC),

and then the IFN-g activated inhibitor of translation (GAIT)

complex is formed. GAIT complex subsequently silences

translation by binding to GAIT elements in the 30-untrans-

lated region of mRNAs that function in pathways for

inflammation. Functional deficiency of GAIT system arising

from genetic mutation or environment stress may aggravate

disorders caused by chronic inflammation [26,27]. At

present, EPRS is found to specifically repress the translation

of proteins such as ceruloplasmin (Cp), vascular endothelial

growth factor A (VEGF-A), death associated protein kinase

(DAPK), chemotactic factors and receptors mediated by GAIT

[27–30]. It is estimated that the EPRS of urine may be released

from dead cells in blood and the urinary tract. EPRS in urine

of T2DM was less than that in healthy controls, which may

show that the reduced expression of EPRS results in an

impairment of protection against the inflammatory re-

sponse, accentuate insulin resistance and/or pancreatic b-

cell dysfunction.

CLU is a conserved disulfide-linked glycoprotein expressed

by a wide array of tissues and is implicated in several

physiologic and pathologic conditions, including diabetes

mellitus. A secreted form displays cytoprotective effects,

whereas a cytoplasmic/nuclear form displays apoptotic

properties [31]. sCLU seems to function as a small heat shock

protein-like chaperon to protect cells from the deleterious

effects of ROS. Normally when cells are stimulated by

increased amounts of ROS, the CLU gene has been shown to

be up-regulated to develop cytoprotective capability. Further-

more, there is growing evidence that clusterin is a growth

factor-like molecule involved in pancreatic b-cell neogenesis

from pancreatic stem cells [32]. Therefore, impaired function

of sCLU may lead to an impairment of protection against

oxidative stress and cause pancreatic b-cell dysfunction [21]. It

is now accepted that resistance to leptin is another important

factor in insulin resistance. Clusterin and leptin complex

retains the ability to transduce the leptin signal, leading leptin

to produce biological effect. Clusterin deficiency will increase

the free form of leptin in the circulation without biological

effect resulting in hyperinsulinemia and insulin resistance

[33]. Therefore, a lower concentration of clusterin is closely

related with insulin resistance and/or pancreatic b-cell

dysfunction.

A large number of biomarkers have been discovered in

urine using MS-based approaches. A deficiency of our study is

that MALDI-TOF MS tends to detect low molecular mass

proteins and peptides ranging from 1000 Da to 20,000 Da and

therefore will not detect peptides of smaller or larger

molecular weight.

Funding

The study was supported by funding from National Key

Technology R&D Program (No. 2013BAI12B01), and funding

from Capital Medical Science and Research (No. 2011-2008-02).

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

We gratefully acknowledge all volunteers for generous

donation of urine samples as well as the staffs in clinical

laboratory of Beijing Shijitan Hospital for enthusiastic assis-

tance.

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