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Inter-individual differences and common features of human urine peptidome

revealed by liquid chromatography-tandem mass spectrometry

Article  in  International Journal of Mass Spectrometry · October 2018

DOI: 10.1016/j.ijms.2018.09.034

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 International Journal of Mass Spectrometry 434 (2018) 272–275

Contents lists available at ScienceDirect

International Journal of Mass Spectrometry

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

Full Length Article

Inter-individual differences and common features of human urine

peptidome revealed by liquid chromatography–tandem mass
spectrometry
S.M. Li a , H. Wang b , X.Y. Hong b , L. Wu b , J.L. Xu b , Y. Wang a,∗
a
College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, China
b
College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology, Shenzhen University, Shenzhen, China

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

Article history: Biomarkers are routinely discovered by comparing the signal intensity of mixture samples from healthy
Received 28 May 2018 and disease, which defaults to the most obvious difference between the two groups are associated with
Received in revised form the disease. Actually, there also may be huge differences among healthy individual samples, so elu-
17 September 2018
cidation of individual differences of healthy samples can help to determine the screening criteria for
Accepted 28 September 2018
biomarkers. The urine sample of a 12 healthy people and 3 mixed urine samples were analyzed by using
Available online 2 October 2018
nano-liquid chromatography-high-resolution tandem mass spectrometry. The number of identified pep-
tides and proteins as well as peptide sequence was compared to explore the compositional features and
Keywords:
Peptidomic
inter-individual differences in the urine peptidome. The numbers of peptides detected ranged from 80
High-resolution tandem mass spectrometry to 1715 per sample, with the number of proteins of origin ranging from 18 to 146. Despite this very
Urine large variance, some peptides were detected in all 15 samples, includes peptides from uromodulin,
Individual difference hemoglobin, fibrinogen, and collagen alpha-2(I) chain. We have found that the results of peptidomics
were more stable at protein level, peptides from proteins such as uromodulin, hemoglobin and fibrino-
gen were detected with high frequency and huge difference of signal intensity in urine of health person,
which suggested that selecting these peptides as disease biomarkers may be compromised by insufficient
specificity.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction to be associated with the same compound for subsequent statisti-

As a complement to proteomics, peptidomics encompasses
the entirety of low-molecular-weight (1–20-kDa) peptides from
biological samples, such as tissues, cells, or body fluids [1]. The
range of molecular weights of peptides included depends on the
methods of separation and identification employed. Traditionally,
surface-enhanced laser desorption ionization-time of flight mass
spectrometry [2,3] and matrix-assisted desorption ionization-time
of flight mass spectrometry (MALDI-TOF-MS) [4,5] have been used
to assess peptidomic differences and to identify possible peptide
biomarkers based on differences in spectral profiles and patterns.
Unfortunately, the resolution of these two technologies is not high
enough to distinguish peptides with extremely close molecular
weights in complex biological samples. Also, sequence informa-
tion cannot be obtained in the absence of tandem MS techniques.
Because of these limitations, close peaks are by default assumed
∗ Corresponding author.
E-mail address: wyong@szu.edu.cn (Y. Wang).
cal analysis [6,7]. High-resolution mass spectrometry and tandem
mass spectrometry techniques can be used to address the afore-
mentioned limitations [8–10]. Use of liquid chromatography couple
with tandem mass spectrometry (LC–MS/MS) is growing in pep-
tidomics research. Besides sequence identification, LC–MS/MS can
be used in quantitative peptidomics, including label-free quanti-
tation and isobaric labeling quantitation (e.g., isobaric tags) for
relative and absolute quantitation (i.e., iTRAQ) [11,12]. Multi-
dissociation methods can also be used to improve peptidome
dataset size [13].
One of the most important applications of peptidomics is the
identification of biomarkers of diseases in bodily fluids, such as
urine. Typically, potential biomarkers are screened for by con-
trasting the peptidomes of a disease group versus a corresponding
healthy group according to the molecular weights of the peptides,
rather than their sequences [14,15]. Hence, there is very little infor-
mation published regarding peptide distribution characteristics at
the protein level, although such information is important for robust
and repeatable biomarker research.

https://doi.org/10.1016/j.ijms.2018.09.034
1387-3806/© 2018 Elsevier B.V. All rights reserved.
 S.M. Li et al. / International Journal of Mass Spectrometry 434 (2018) 272–275 273

Table 1
Total numbers of peptides and proteins found in 15 samples and their distributional features.

Sample No. peptides No. proteins No. (%) peptides from No. (%) proteins represented by
top-10 proteinsa a single peptide identified

Individual samples
1 80 18 34 (43%) 11 (61%)
2 85 29 54 (64%) 14 (48%)
3 85 37 38 (45%) 24 (67%)
4 107 36 44 (41%) 20 (56%)
5 139 37 86 (62%) 23 (62%)
6 155 35 106 (68%) 22 (63%)
7 202 50 73 (30%) 33 (66%)
8 225 62 118 (52%) 30 (48%)
9 267 55 115 (44%) 21 (38%)
10 567 108 241 (42%) 43 (39%)
11 1486 122 899 (60%) 51 (44%)
12 1715 146 1033 (60%) 46 (32%)
Mixed samples
13 354 77 102 (29%) 34 (44%)
14 425 68 225 (53%) 26 (38%)
15 455 82 279 (61%) 39 (48%)
a
The top 10 proteins are listed in Table 2.

Previously, we have identified 790 peptides originating from
125 proteins in a single urine sample using high-resolution tan-
dem mass spectrometry [16]. The aims of the present study were,
first, to explore the data volume and common features of the human
urine peptidome and, second, to compare the urine peptidomes of
healthy individuals based on the analysis of 12 individual samples
and 3 mixed age-band samples.
2. Experimental
2.1. Subjects
Fifty-seven participants were recruited from Shen Zhen Univer-
sity.
2.2. Instruments and reagents
The following instruments were employed in this work:
EksigentnanoLC-UltraTM 2D-LC (AB SCIEX, USA); TripleTOF 5600
high resolution mass spectrometer (AB SCIEX, USA) with Protein
Pilot 4.5 software (AB SCIEX, USA); vacuum freeze-drying machine
(Thermo savant, USA); and an ultra-pure water purifier (Thermo
scientific, USA). Graphene oxide-lanthanum nano-magnetic com-
posites were used with RP mobile phase A (98% water, 0.1% formic
acid, and 2% acetonitrile) and RP mobile phase A (2% water, 0.1%
formic acid, 98% acetonitrile).
2.3. Peptide separation and enrichment
Urine samples (N = 57) were collected from all participants,
o
placed on ice, and then stored at −80 C after packing. Of the 57 sam-
ples, 45 were used for mixed samples wherein each of three mixed
samples was produced by combining samples from 15 individuals.
The remaining 12 individual samples were analyzed separately.
A 1.5-ml aliquot of urine was taken from each sample for pep-
tide separation and enrichment. The samples were centrifuged
for 10 min at 10,000 r/min for impurity removal; then 30 ␮l
of 30 mg/ml oxide-lanthanum nano-magnetic composites were
added to the supernatants. Subsequently, the samples were vor-
texed for 2 min, shaken for 10 min at room temperature, and then
centrifuged for 5 min at 10,000 r/min. After centrifugation, the sam-
ples were subjected to magnetic separation. Supernatants were
removed and the precipitates were washed with water three times,
eluted with 20 ␮l of 80% acetonitrile-1% trifluoroacetic acid solution
twice, and then lyophilized.
2.4. Biochemical analysis
The lyophilized peptide samples were re-dissolved in 98% water,
2% acetonitrile, and 0.1% formic acid solution. Online nano-RPLC
was performed in a liquid chromatography EksigentnanoLC-
UltraTM 2D system. After each sample was dissolved, it was
pre-loaded onto the C18 trapping column at a rate of 2 ␮L/min,
onto the C18 column (100 ␮m × 3 cm, C18, 3 ␮m, 150 Å; Eksigent,
USA), then desalinated for 10 min. The samples were analyzed in
a C18 reverse-phase column (75 ␮m × 15 cm, C18, 3 ␮m, 120 Å;
Eksigent, USA) with the following experimental gradient: minute
0–42, 5–25% B; minute 42–56, 25–40% B; minute 56–64, 80% B;
and minute 64–70, 5% B. MS was performed with a TripleTOF 5600
system and binding nanospray III ion source (AB SCIEX). The spray
voltage was 2.4 kV, the curtain air pressure was 30 psi, the atom-
ization gas pressure was 5 psi, and the heating temperature 150 ◦ C.
The scan time for a single TOF-MS was 250 ms. IDA cycles collected
up to 35 secondary patterns (charges in the range of 2+ ∼ 8+, count
>100 per second, the cumulative time for each secondary pattern
is 80 ms. The cycle time was set to 2.5 s, the collision cell energy set
was suitable for the full range of precursor ion collision-induced
dissociation, and dynamic exclusion was set to 11 s.
2.5. Data analysis
Original wiff files were processed in Protein Pilot Software
v. 4.5 (AB SCIEX, USA) with a Homo sapiens-specific uniprotli-
brary database (containing 20,210 protein sequences, downloaded
January 2, 2015), search parameters were non digestion, phospho-
rylation and biological modification, retrieval mode is thorough
analysis, the false positive rate is controlled to 1% FDR.
3. Results and discussion
3.1. Data size of 12 individual urine samples and 3 mixed urine
samples
The quantity of peptides detected in each of the 12 individual
samples ranged from 80 to 1715 (Table 1), with three samples hav-
ing fewer than 100, three having 100–200, three having 200–300,
and two samples having >1400 peptides. Although the number
of corresponding proteins increased with the number of detected
peptides, the two values were not strictly correlated. For exam-
ple, 85 peptides were detected in both sample 2 and sample 3, but
arising from 29 and 37 proteins, respectively. In urine sample 6,
274 S.M. Li et al. / International Journal of Mass Spectrometry 434 (2018) 272–275

Table 2
The 10 highest frequency (Freq.) proteins and the number of detected peptides and characteristic peptide sequence associated with each of them.

Rank Protein Freq. Average no. derived peptides per sample Characteristic sequence

1 Uromodulin 14/15 26 SGSVIDQSRVLNLGPITR

2 Hemoglobin ␣ subunit 12/15 29 AAHLPAEFTPAVHASLDKFLASVSTVL
3 Collagen ␣-2(I) chain 13/15 12 AGPPGAPGAPGAPGPVGPAGKSGDRGETGP
4 Hemoglobin ␤ subunit 12/15 29 YQKVVAGVANALAHKY
5 Fibrinogen ␣ chain 13/15 12 TGNRNPGSSGTGGTATWKPGSSGP
6 Fibrinogen ␤ chain 14/15 5 DKKREEAPSLRPAPPPISGGGY
7 ␣-2-HS-glycoprotein 11/15 10 MGVVSLGSPSGEVSHPRKT
8 Isoform 2 of clusterin 11/15 10 ASHTSDSDVPSGVTEVVVKLFDSDPITVTVPVEV
9 Kininogen-1 13/15 4 KRPPGFSPFR
10 Major prion protein 12/15 4 NTGGSRYPGQGSPGGNRYPPQGGGG

155 detected peptides were found to have arisen from only 35 pro- Table 3
Comparison of highly abundant proteins (top 15) in peptidome and proteome
teins. The peptide:protein number ratio differed across different
obtained for the same urine sample from a single individual.
samples. In samples 1–10, the ratio was between 3 and 5, with an
average value of about 4, whereas in samples 11 and 12 the average Peptidome
peptide:protein ratios were about 12. In the three mixed samples Protein No. peptides
(samples 13, 14 and 15), the numbers of peptides were found to
␣-1-antitrypsin 490
lie between 300 and 400 and the numbers of proteins were in the Apolipoprotein A-I 90
range of 60–70. Leucine-rich ␣-2-glycoprotein 56
The distribution of peptides at the protein level was uneven. The Corticosteroid-binding globulin 44
ten most frequently associated proteins accounted for the greatest Secreted and transmembrane protein 1 40
Plasma protease C1 inhibitor 39
proportions of detected peptides in all 15 experimental samples. ␣-1-antichymotrypsin 37
The percentage of these top-10 proteins in each sample ranged Transthyretin kininogen-1 32
from 29% to 68% (average, 51%). These findings suggest that a large ␣-1B-glycoprotein 31
portion, perhaps almost half, of the detected peptides were derived Isoform 2 of clusterin 35
Vesicular integral-membrane protein VIP36 32
from the top-10 proteins in the urine peptidome. On the other hand,
Angiotensinogen 26
some proteins (mean, 50% of peptides/individual sample; range, ␣-2-HS-glycoprotein 20
32–67%) were represented by only a single peptide in the urine Thyroxine-binding globulin 28
peptidome. Hemoglobin ␣ subunit 23
The largest identified peptide in the urine peptidome
(7072.634 Da) was a segment of the ␤ subunit of hemoglobin. Proteome

Its sequence was DEVGGEALGRLLVVYPWTQRFFESFGDLSTPDA Protein No. peptides

VMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFA. In addition, differ- Serum albumin 566
ent from the results of Fricker [17], we were able to detect peptides ␣-1-antitrypsin 178
containing a cysteine with a free-SH (e.g., TVVQPS VGAAAGPVVP- Serotransferrin 84
PCPGRIRHF) in 6 of the 15 samples. Prostaglandin-H2 D-isomerase 51
Uromodulin 46
␣-1B-glycoprotein 54
3.2. Common features of the urine peptidome at the protein level Haptoglobin 26
Ceruloplasmin 24
Ig lambda-2 chain C regions 31
Although relative quantitative analysis of peptides in urine is Gelsolin 23
widely used to identify potential disease biomarkers, to our knowl- Low-density lipoprotein receptor-related protein 2 17
edge, it has not been reported whether there are any common Ig kappa chain V-III region HIC 29
features of urine peptidomes. When we analyzed the peptidomic Clusterin 23
Galectin-3-binding protein 16
data on a protein level, we found that the degradation products
Ig ␣-1 chain C region 34
of some proteins were detected with particularly high frequen-
cies. As shown in Table 2, the most frequently detected protein
was uromodulin, which was detected in 14 of the 15 samples.
proteins [18]. To examine the relationship between the urine pep-
Peptides arising from hemoglobin ␣ and ␤ subunits, fibrinogen ␣
tidome and urine proteome, we performed a proteomic analysis of
and ␤ chains, the collagen alpha-2(I) chain, and ␣-2-HS glycopro-
sample 12, the sample in which the largest number of peptides was
tein were also detected frequently (detection frequencies ≥73.3%).
identified and compared the results with our peptidome results. In
Unlike findings from proteomics studies, the detection frequencies
urine peptidome of sample 12, we identified 1715 peptides derived
of these proteins did not correlate directly with the average number
from 146 proteins.
of associated peptides that were detected. For example, although
In the proteome sample 12, after enzyme trypsin digestion, we
kininogen-1 and hemoglobin ␣ subunit were detected in a similar
detected 2345 peptides from 221 proteins. The 15 most abundant
number of samples (13 and 12, respectively), the average number
proteins in the urine peptidome and proteome and the num-
of detected peptides arising from the former was 4 whereas that
bers of detected peptides in each are reported in Table 3. Only
from the latter was 29.
two proteins were among the 15 most abundent in both the
peptidome and proteome: ␣-1-antitrypsin and ␣-1B-glycoprotein.
3.3. Differences between the urine peptidome and proteome Serum albumin, the most abundent protein in the urine proteome,
was also found in the urine peptidome, but at low levels. Con-
In a cell peptidomics study, Gelman et al. found that most of pep- versely, the angiotensinogen degradation peptides transthyretin
tides found in the cell lines examined were not derived from the and kininogen-1 were detected only in the urine peptidome. The
most abundant cellular proteins nor from the most unstable cellular 146 proteins in the peptidome and the 221 proteins in proteome
 S.M. Li et al. / International Journal of Mass Spectrometry 434 (2018) 272–275
included 53 shared proteins. Thus, the two datasets are largely dis-
tinct, albeit with a fair overlap. Such comparative analyses may be
a useful approach in biomarker research.
It has been suggested that potential biomarkers may be iden-
tified based on upregulation or downregulation of mass peaks
in MALDI-TOF spectra [19]. However, because of the ionization
suppression effect that occurs with complex samples such as
urine, far fewer peptides are detected by MALDI-TOF/MS than by
LC–MS/MS, limiting the number of peptides that MALDI-TOF/MS
may yield [3,6,15]. LC–MS/MS can provide a more accurate and
robust description of possible biomarkers from the perspective
of protein degradation, and also more information about peptide
sequences. Furthermore, the molecular weights of some peptides
that are too close to be distinguished by MALDI-TOF/MS can be
identified by LC–MS/MS. For example, two similarly sized pep-
tides derived from ␣-2-HS-glycoprotein (2490.227 Da) and the
hemoglobin ␣ subunit (2490.268 Da) (LAAPPGHQLHRAHYDLRHTF
and HLPAEFTPAVHASLDKFLASVS, respectively), have elution times
of 51 min and 36 min, respectively.
Using LC–MS/MS, we found very large inter-individual dif-
ferences in urine peptidome dataset size. Because all of the
procedures, including sample collection, volume, and analysis,
were the same for all of the samples, this broad range in the num-
ber of peptides found in the samples (from 80 to 1715 peptides)
indicates that the urine peptidome is diverse. In other words, it
is normal to obtain different peptidomics results from different
samples, even when all of them came from presumably healthy
people. Hence, much inter-individual urine peptidome variance
appears to be inevitable and not indicative of any abnormal-
ity. The distribution of peptides on a protein level was relatively
more stable, with peptides that are degradation products of uro-
modulin, hemoglobin, fibrinogen, and ␣-2-HS-glycoprotein being
detected in more than 90% of the samples. Hence, the degradation
products of these proteins in urine should be considered nor-
mal.
Given these results, we caution against using high-frequency
peptides as biomarkers due to the risk of insufficient specificity. For
example, a degradation product of uromodulin (peptide SGSVIDQS-
RVLNLGPITR) that emerged as a basic component of the urine
peptidome in this study (Table 3) was proposed previously to be
considered a potential biomarker of major depressive disorder [15],
preeclampsia [20], acute rejection of renal transplantation [21], and
systemic juvenile idiopathic arthritis [22]. However, the ubiquitous
presence of this peptide in urine makes it likely to be detected at
varying levels across individuals and thus susceptible to generating
type II errors.
Furthermore, we believe that the urine peptidome can be
divided into physiology-related and disease-related components.
That is to say, we expect that some peptidome differences reflect
the particular physiological metabolism of individuals. Further-
more, peptides derived from disease-related proteins should be
detected by way of qualitative analysis, which is more robust and
reliable than quantitative analysis.
4. Conclusions
We observed a very large inter-individual variance in urine pep-
tidomics composition in healthy urine samples, which complicates
efforts to identify disease biomarkers with peptidomic analysis.
Efforts to identify biomarkers of pathophysiology in urine should
focus on critical changes, such as the degradation, in disease-related
proteins.
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275
Acknowledgment
This work was supported by theBasic Research Project of Shen-
zhen (No. JCYJ20170818142154551).
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