comprehensive analysis of low-abundance proteins in human urinary exosomes using peptide ligand...

$: Comprehensive analysis of low-abundance proteins in human urinary exosomes using peptide ligand library technology, peptide OFFGEL fractionation and nanoHPLC-chip-MS/MS$
Research Article

Comprehensive analysis of low-abundanceproteins in human urinary exosomesusing peptide ligand library technology,peptide OFFGEL fractionationand nanoHPLC-chip-MS/MS

Human urinary exosomes are 30–100 nm vesicles that originate as the internal vesicles

in multivesicular bodies from every renal epithelial cell type facing the urinary track and

may serve as a suitable noninvasive starting material for biomarker discovery relevant to

a variety of renal disease. To comprehensively explore the low-abundance proteome,

combinatorial peptide ligand libraries, combined with peptide OFFGEL electrophoresis

were employed for the enrichment and separation of relatively low-abundant proteins in

urinary exosomes. After analysis by nanoHPLC-chip-MS/MS, 512 proteins were identi-

fied, including a large number of proteins with extreme molecular weight or extreme pIvalue, which could not be well mapped by using traditional 2-D-gel-based separation

methods. This in-depth analysis of low-abundant proteins in urinary exosomes led to an

increased understanding of molecular composition of these little vesicles and may be

helpful for the discovery of novel biomarker. Our work also provides an effective strategy

of concentration and identification of low-abundance proteome from complex

bio-samples.

Keywords:

Combinatorial peptide ligand libraries / OFFGEL electrophoresis / Proteome /Urinary exosomes DOI 10.1002/elps.201000401

1 Introduction

Human urinary exosomes are 30–100 nm vesicles that

originate as the internal vesicles in multivesicular bodies

from every renal epithelial cell type facing the urinary track.

Of the total urinary proteins excreted, 3% were in exosomes

[1]. Urinary exosomes contain proteins that are character-

istic of every renal tubule epithelial cell type, as well as

podocytes and transitional epithelia from the urinary

collecting system [2]. Therefore, urinary exosomes may

provide a suitable noninvasive starting material for biomar-

ker discovery relevant to a variety of renal disease processes

because of its convenience of collection and the ability of

being collected repeatedly over lengthy time periods. Mean-

while, prefractionation of exosomes from urine can be

useful as a means of enriching for markers of particular

types of disease.

There have been many studies focusing on the physio-

logical and pathophysiological significance of urinary

exosomes, and several exosome-associated candidate

proteins of potential diagnostic value have been identified.

Aquaporin-2 (AQP2), present in these vesicles, appears to

correlate with circulating vasopressin levels, and measure-

ments of its excretion have begun to be exploited for the

study of water balance abnormalities in humans [3, 4].

Pisitkun et al. demonstrated the presence of multiple

protein products of genes in urinary exosomes known to be

responsible for renal and systemic diseases, including

autosomal dominant polycystic kidney disease, Gitelman

syndrome, Bartter syndrome, autosomal recessive syndrome

of osteopetrosis with renal tubular acidosis, and familial

renal hypomagnesemia [5]. According to Zhou et al. [6],

urinary exosomal Fetuin-A might be a predictive biomarker

of structural renal injury, as it is elevated in patients with

acute kidney injury, but not in prerenal azotemia. A

previous study found that urinary Na1/H1 exchanger

isoform 3 (NHE3), a typical membrane protein, increases in

patients with acute renal failure [7]. Thus, urinary exosomal

Yuan Zhang1

Yanyan Li1

Feng Qiu2

Zongyin Qiu1

1College of Pharmacy,Chongqing Medical University,Chongqing, P. R. China

2First Affiliated Hospital,Chongqing Medical University,Chongqing, P. R. China

Received August 2, 2010Revised August 29, 2010Accepted September 15, 2010

Colour Online: See the article online to view Figs. 1–3 in colour.

Abbreviations: AQP2, aquaporin-2; CPLL, combinatorialpeptide ligand library; GO, gene ontology; MW, molecularweight; OGE, OFFGEL electrophoresis; THP, Tamm–Horsfallprotein

Correspondence: Professor Zongyin Qiu, College of Pharmacy,Chongqing Medical University, Chongqing, 400016, P. R. ChinaE-mail: [email protected]: 186-023-68485277

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2010, 31, 3797–3807 3797

proteomics may provide an avenue for the discovery of

urinary biomarkers useful for early detection of kidney

diseases and for monitoring of treatment [8].

Because of the large variety of species of proteins and

peptides and their broad dynamic range of concentrations,

the sample prefractionation steps conducted prior to mass

detection are critically important for the comprehensive

analysis of complex biosamples such as plasma, serum and

urine. In proteomic researches, to achieve high resolution of

proteins especially low-abundance proteins, it is crucial to

establish effective high-abundance proteins depletion

methods and sample fractionation methods. Various studies

have suggested that low-abundance proteins are the most

important resource of discovering candidate biomarkers in

biosamples [9, 10].

To date, the most commonly used exosomes isolation

method is high-speed centrifugation, but this procedure is

usually interfered by Tamm–Horsfall protein (THP). THP,

also known as uromodulin, is the most abundant protein in

human urine. THP is found in the urine as a disulfide

bonds linked high-molecular-weight polymer assembled

into filaments or matrices. When the urine sample is

centrifuged at 200 000� g, a large amount of these polymers

pellet along with exosomes and become the high-abundant

protein in exosomal proteome. And also because THP can

entrap other proteins, the presence of large amounts of THP

and other highly abundant proteins in urinary exosomes

masks the identification of other less abundant proteins. In

a recent work, Gonzales et al. mixed the resuspended pellet

with 200 mg/mL DTT at 951C for 2 min to disrupt the

polymeric network by reduction of disulfide bonds linking

the monomers in the THP [11]. However, by using this

method, other highly abundant proteins may not be elimi-

nated. Here, we used the technology of combinatorial

peptide ligand library (CPLL) to reduce the dynamic range of

protein concentration and tried to unmask previously

undetected proteins in urinary exosomes. This method is

based on the treatment of sample with the combinatorial

libraries of hexameric peptide ligands bound to porous

polyacrylate beads. Each bead contains billions of copies of a

unique hexapeptide ligand distributed throughout its porous

structure. And every bead potentially has a ligand different

from every other bead. Therefore, with a population of

millions of individual peptide ligands obtained by combi-

natorial chemistry, any protein in the starting material can

theoretically interact with one or a few beads among the

wide diversity (10–206) of ligand beads from the library.

Once the most abundant protein species have saturated

their binding sites, the remaining molecules are washed

away in the flow-through, while minor protein species are

progressively enriched on their corresponding beads. The

protein mixture is thus ‘‘equalized’’, and the dynamic range

of protein concentrations is strongly reduced [12]. This

ligand library has been efficiently applied to capture and

reveal a very large population of previously undetected

proteins in several types of samples such as urine [13],

serum [14], platelets [15] and red blood cells [16]. Compared

with the commonly used antibody-based depletion method,

CPLL can avoid the high dependence on antibodies and a

series of limitations, such as co-depletion of associated

species and the dilution of the collected, depleted sample

[17, 18].

Reducing sample complexity through efficient fractio-

nation also allows more complete in-depth analysis of the

sample with MS/MS. In the present research, after deple-

tion of highly abundant proteins, OFFGEL electrophoresis

(OGE) was introduced to separate low-abundance proteins

in urinary exosomes. IEF, a high-resolution electrophoresis

technique for separation and concentration of peptides and

proteins based on their pI, has been widely used in

proteomic research. However, the tedious post-IEF sample

processing of traditional IPG-based IEF is a major limitation

of the method, which may lead to the loss of important

proteins or dilution of the sample. OGE is a recent advance

in separation technology that fractionates proteins or

peptides according to their pI [19–22]. This technique

achieves the same high resolution as IPG gels but simplifies

the sample processing by recovering the separated peptides

or proteins in liquid phase, which makes it much more

convenient for subsequent LC-MS/MS analysis and elim-

inates the need for tedious and error-prone peptide isolation

from the IPG gel. Furthermore, OGE applied to predigested

peptides has been shown to be an excellent alternative to ion

exchange chromatography in shotgun proteomics, since the

theoretical pI value of the peptide can be used as an inde-

pendent validating and filtering tool during database search

for MS/MS peptide sequence identification, which increases

the reliability of protein identification procedures [23]. Thus,

this technique was demonstrated to be of great interest in

shotgun proteomics.

In the present research, the CPLL-treated urinary

exosomes were predigested and fractionated by peptide OGE

and characterized by mass detection of nanoHPLC-chip-

MS/MS analysis. By using this strategy, 512 unique proteins

were identified from the human urinary exosomes, includ-

ing a large number of highly basic proteins and proteins

with extreme molecular weight (MW). Gene ontology (GO)

analysis was also employed to analyze the biological process,

molecular function and cellular component of the low-

abundance proteome of urinary exosomes.

2 Materials and methods

2.1 Urine collection

Pooled human mid-stream urine specimens (first urine in

the morning) were collected from eight normal individuals

(four males and four females, ages in the range of 23–28),

who underwent a medical checkup and had not consumed

aspirin or other nonsteroidal anti-inflammatory drugs for at

least 2 weeks. All females had no menstrual cycle at the time

of collection. The urine samples were collected in the

polypropylene centrifuge tubes. Fifty milliliters per subject

Electrophoresis 2010, 31, 3797–38073798 Y. Zhang et al.


was collected and mixed together. Immediately after urine

collection, the protease inhibitors were added (1.67 mL of

100 mM NaN3, 2.5 mL of 11.5 mM PMSF and 50 mL of

1 mM leupeptin) to avoid proteolysis. The urine samples

were stored on ice prior to centrifugation at 1000� g for

15 min at 41C. The precipitates were removed and the

supernatants were stored at 201C until use. The protein

concentration of the urine samples was measured using the

Bradford Assay.

2.2 Exosomes isolation

The mixed sample was centrifuged at 17 000� g for 15 min

at 41C. The 17 000� g supernatant was ultracentrifuged at

200 000� g for 1 h at 251C in a Hitachi Refrigerated

Centrifuge (Hitachi, Tokyo, Japan). And the ultracentrifuga-

tion step was repeated two additional times, adding new

17 000� g supernatant volume each time. The final pellet of

urinary vesicles was pooled together, suspended in 150 mL of

PBS, aliquoted and stored at �801C [5].

2.3 Immunoelectron microscopy of immunogold

labeled exosomes

Vesicle suspensions were cross-linked using 4% parafor-

maldehyde/0.25% glutaraldehyde in PBS for 1 h, dehydrated

in graded ethanol and cut into ultra-thin sections

(70–80 nm). Sections were then applied to 200-mesh nickel

grids. After blocking with 5% BSA and washing, the grids

were incubated with primary antibody solutions (1:50–1:500

dilutions) for rabbit polyclonal antibody to AQP2 (Beijing

Boisynthesis Biotechnology, Beijing, China) for 2 h at room

temperature. Following PBS rinses, the grids were then

exposed to species-specific anti-IgG antibodies conjugated to

colloidal gold particles (5 nm) (Beijing Boisynthesis Biotech-

nology). After washing, membranes underwent negative

staining with 0.5% uranyl acetate. Finally, the grids were

examined with a PHILIPS-TECNAI 10 electron microscope

(Philips, Holland) operated at 80 kV after drying. Control

labeling was performed identically, but nonimmune IgG

was substituted for the primary antibody.

2.4 Sample treatment with CPLLs

Urinary exosomes sample was loaded onto a column of

100 mL peptide library beads (Bio-Rad, Hercules, CA, USA).

To insure effective binding, the column was slowly rotated

for 2 h prior to centrifugation at 1000� g for 60 s to get the

unbounded fraction (flow-through fraction). Then, the

column was washed with 200 mL of 25 mM of phosphate

buffer, pH 7.4, and repeated this two more times (wash

fraction). The adsorbed proteins were eluted sequentially

with the following different solutions: (i) three consecutive

times with 20 mL of an acidic Urea/CHAPS buffer (5% acetic

acid, 8 M urea and 2% CHAPS) (E1 fraction); (ii) three

consecutive times with 20 mL of 10% SDS – 3% DTT, boiled

for 5 min and the protein recovery solution was spun at

1000� g and collected (E2 fraction). The E1 and E2 fractions

were desalted through a 3 kDa cut-off centrifugal tube

(Millipore, Billerica, MA, USA).

2.5 1-D SDS-PAGE

Twenty microliters of each elution, flow-through and wash

fractions from the library column, as well as 20 mL of the

initial nontreated human exosomes isolation were diluted in

Laemmli buffer and boiled for 5 min before being separated

on a 12% acrylamide SDS-PAGE gel. Proteins were

visualized by Coomassie Blue staining.

2.6 Tryptic in-solution digestion

The CPLL-treated sample was reduced and denatured using

50% 2,2,2-trifluoroethanol with 200 mM DTT at 951C for

20 min, followed by alkylation with iodoacetamide at room

temperature for 1 h. The reduced and alkylated sample was

diluted 1:10. Trypsin (Promega, Madison, WI, USA) was

added at 1:20 enzyme: substrate. And then, the sample was

incubated overnight at 371C.

2.7 Peptide OGE

For the pI-based peptide separation, the 3100 OFFGEL

fractionator and the OFFGEL kit pH 3–10 (Agilent

Technologies, Palo Alto, CA, USA) with a 12-well setup

was used according to the protocol of the manufactory. Ten

minutes prior to sample loading, 12-cm-long IPG gel strips

with a linear pH gradient ranging from 3 to 10 were

rehydrated in the assembled device with 25 mL of focusing

buffer composed of 7 M urea, 2 M thiourea, 1% w/v DTT

and 0.5% v/v ampholytes pH 3.0–10.0 per well [24]. About

200 mg of peptide sample was diluted in focusing buffer, and

the sample was loaded in each well. The sample was focused

at typical voltages ranging from 500 to 4000 V until 50 kVh

was reached after 24 h, with a maximum current of 50 mA

per strip. After the peptide OGE, the recovered fractions

(volumes between 50 and 150 mL) were lyophillized and

stored at �801C until use.

2.8 LC-MS/MS analysis

Peptides were resuspended in 25 mL 0.1% formic acid and

20 mL was used for each LC-MS/MS analysis. An Agilent

1200 series Nanoflow HPLC system (Agilent Technologies)

was run in the trapping mode with an enrichment column

(560.3 mm, 5 mm particles) and a Zorbax 300SB C18

analytical column (150� 0.075 mm, 3.5 mm particles).

Electrophoresis 2010, 31, 3797–3807 Proteomics and 2-DE 3799


Sample was injected on the enrichment column via an

autosampler. The mobile phase consisted of solvents A

(water with 0.1% formic acid) and B (90% ACN, 10% water

with 0.1% formic acid). The column was developed with a

biphasic gradient of solvent B from 3 to 15% in solvent A in

2 min followed by an increase of B from 15 to 50% in

70 min. The column was regenerated by ten column

volumes of 90% B followed by five volumes of 3% B. Both

the enrichment and the analytical columns were submitted

to the same development, washing and regeneration

conditions. The total analysis time was 120 min, and the

flow rate was fixed at 0.3 mL/min. MS analysis was

conducted on an Agilent 1100 Series LC/MSD Trap MS

equipped with an Agilent orthogonal nanoelectrospray

source with data-dependent MS/MS acquisition. The MS

and MS/MS conditions employed were:

Drying gas flow: 4 L/min, 3251C; capillary voltage:

1900 V; skim 1: 30 V; capillary exit: 75 V; trap drive: 85;

averages: 1; ion current control: on; maximum accumula-

tion time: 150 ms; smart target: 500 000; MS scan range:

300–2200; ultra scan: on.

MS/MS: number of parents: 5; averages: 1; fragmenta-

tion amplitude: 1.3 V; SmartFrag: on, 30–200%; active

exclusion: on, 2 spectra, 1 min; prefer 12: on; exclude 11:

on, MS/MS scan range: 200–2000; ultra scan: on; ion

current control target: 500 000.

Due to statistical fluctuations of peptide precursor

selection during MS/MS acquisition, three LC-MS/MS

assays were run with each sample in order to be able to do a

proper proteome comparison.

2.9 Protein identification and data analysis

Peptide and protein identifications were run automatically

with the Spectrum Mill Proteomics Workbench Rev

A.03.03.078 software from Agilent Technologies. Peak lists

were created with the Spectrum Mill data extractor program

with the following parameters: scans with the same

precursor71.4 m/z were merged within a time frame of

715 s; precursor ions needed to have a minimum S/N of

25; charges up to a maximum of 7 were assigned to the

precursor ion and the 12C peak was also determined by the

Data Extractor; MH1 comprised between 450 and 4000, and

a scan time comprised between 0 and 300 min was used.

Peptides were automatically identified by the Spectrum Mill

Proteomics Workbench Rev A.03.03.078 software using

UniProtKB/Swiss-Prot protein database (Geneva, Switzer-

land) search for tryptic peptides with the restriction to Homo

Figure 1. Immunoelectron microscopy of urinary exosomes.Urinary exosomes separated from normal human urine weredemonstrated by immunogold electron microscopy of urinarylow-density membranes probed with antibody to water channelAQP2. The red arrows indicate gold particles (5 nm in diameter)labeled on exosomes. The bar denotes 100 nm.

Figure 2. Treatment of urinary exosomes on peptide ligandlibraries and nanoLC-MS/MS analysis. (A) Analysis of proteinfractions by mono-dimensional SDS-PAGE. The four left lanes ofthe electrophoresis image represent molecular mass markers(M), the initial nontreated urinary exosomes (N), the columnflow-through (F) and the phosphate buffer-washed fraction (W).E1 and E2 fractions represent eluted proteins from the peptidelibraries by means of an acidic Urea/CHAPS buffer, and a boilingSDS–DTT buffer. (B) Venn diagrams giving the extent of overlapof proteins identified by nanoLC-MS/MS analysis betweennontreated and CPLL-treated sample.



sapiens. A mass tolerance of 72.5 Da for the precursor ions

and a tolerance of 70.7 Da for the fragment ions were used.

Two missed cleavages were allowed. Carboxymethylated

cysteines were set as fixed modification and oxidized

methionine as variable modification. Spectrum Mill scores

above 13 for complete proteins with a minimum score of 7

and scored peak intensity of 70% for 21, 31 and 41

precursor ions of individual peptides were used as auto-

validation criteria. These criteria are a good compromise

between risking too many false positive if the values are set

too low and the risk to miss real protein hits if the values are

set too high. Auto-validation was necessary to ensure the

same data analysis conditions for all the LC-MS/MS runs

done in this work and to assure a fair comparison of the

results.

2.10 GO analysis

GO analysis was completed using the Generic GO Term

Finder [25] developed by the Bioinformatics Group at the

Lewis–Sigler Institute at Princeton (http://go.princeton.

edu). The objective of GO is to provide controlled vocabularies

for the description of the biological process, molecular function

and cellular component of gene products.

3 Results and discussion

3.1 Exosomes isolation and identification

For proteomic research, it is necessary to get purified

exosomes. To date, the most commonly used exosomes

isolation methods are high-speed centrifugation and ultrafil-

tration. In this research, we employed differential ultracen-

trifugation to purify urinary exosomes. To verify whether the

sediments separated from normal human urine were

exosomes, the vesicles were concentrated and visualized by

immunogold electron microscopy using antibodies reacting

with water channel AQP2. Urinary exosomes were defined on

the basis of the size (30–100 nm) and the shape (round) of the

vesicles. As is shown in Fig. 1, analyzed by TEM, purified

exosomes are relatively round vesicles ranging from approxi-

mately 30–100 nm in diameter. The expression of AQP2 on

the vesicles also argues positively for the presence of exosomes,

as has been previously reported in human urine samples [5].

Figure 3. MS/MS spectra of three peptides derived from low-density lipoprotein receptor-related protein 2 precursor. (A) MS/MSspectrum of a peptide, which was separated into fraction 1 by OGE. The spectrum shows the fragmentation pattern of a doubly chargedprecursor ion at m/z 642.17, pI 4.21. Interpretation of the complete y-ion and b-ion series provides the peptide sequence(R)IDMVNLDGSYR(V) as shown. (B) MS/MS spectrum of a peptide, which was separated into fraction 5 by OGE. The spectrum showsthe fragmentation pattern of a doubly charged precursor ion at m/z 1306.4, pI 3.91. Interpretation of the complete y-ion and b-ion seriesprovides the peptide sequence (R)GIAVDPTVGYLFFSDWESLSGEPK(L) as shown. (C) MS/MS spectrum of a peptide, which wasseparated into fraction 7 by OGE. The spectrum shows the fragmentation pattern of a triply charged precursor ion at m/z 572.45, pI 5.33.Interpretation of the complete y-ion and b-ion series provides the peptide sequence (R)HLCHCEEGYILER(G) as shown.



3.2 Sample treatment with CPLL

As shown in Fig. 2(A), two very intense bands (about 55 and

100 kDa) were observed in the lane of initial nontreated

urinary exosomes (lane Nontreated ‘‘N’’). The most abun-

dant protein centered at about 90 kDa was identified to be

THP, and its presence can interfere with the detection of

other proteins if it is not removed [5]. To increase the

identification of relatively low-abundance proteins in the

region previously occupied by THP and other high-

abundance proteins in the gel, CPLL was employed to

reduce the dynamic concentration range of the

sample and obtain access to the identification of minor

species.

In our experiment, a total amount of 2.1 mg of proteins

was loaded on the combinatorial peptide ligand column, and

the captured proteins were collected by eluting the column

sequentially with an acidic Urea/CHAPS buffer and a boil-

ing SDS-DTT buffer. From the two resulting eluates, 220

and 26 mg of proteins were collected, representing about 10.5

and 1.2% of the initial input. The two different elution

systems had very distinct recovery rate. With the acidic

Urea/CHAPS buffer, most captured proteins were eluted.

To recover more proteins from the beads, a boiling SDS-

DTT buffer, which was more powerful, was sequentially

employed to elute almost quantitatively the adsorbed

proteins [26]. 1-D SDS-PAGE analysis of all fractions under

reducing conditions revealed different patterns of proteins.

After treatment with CPLL, very different patterns were

obtained for the column eluates, which showed a large

decrease of the intense bands, and the apparition of

many new protein bands. As shown in Fig. 2(A), the most

abundant band, at about 90 kDa (lane Nontreated ‘‘N’’ and

flow-through ‘‘F’’), was considerably diminished in the

eluate bands (lane E1 and E2), whereas many more bands

were manifested compared to the nontreated sample,

Figure 4. Analysis of low-abundanceproteome in urinary exosomes by OGEand nanoHPLC-Chip-MS/MS. About 200 mgCPLL-treated urinary exosomes were frac-tionated into 12 wells, and each 20 mL perwell was further analyzed by nanoHPLC-Chip-MS/MS. The number of proteins andpeptides identified in each fraction is illu-strated.



covering a large mass interval from 10 to 120 kDa.

Compared with the THP depletion method of adding DTT

to the sample, the CPLL technology showed more advan-

tages in depletion of all types of high-abundance proteins,

not only the THP.

3.3 Peptide OGE

It has been reported that the analysis of the predigested

peptides by OGE leads to the increased number of identified

peptides and improvement of ability of MS search against

the database [27]. In the present research, peptide OGE was

introduced to separate tryptic-digested peptides after treat-

ment with CPLL using a 12-wells device encompassing the

pH range 3–10. After fractionation and acidification, a

portion (20 mL per well) of the sample was injected directly

onto a chip-based reversed-phase column without additional

sample preparation.

By using peptide OGE, 4730 peptides corresponding to

1688 proteins were identified in 12 fractions of 200 mg CPLL-

treated peptides, leading to the identification of 512 unique

proteins totally. As several peptides with different pI were

fractionated into separate wells, a protein can be identified

from several separate wells, which increases the reliability of

protein identification procedures. As is in this research, 159

out of 512 (31%) proteins were identified in at least two

wells. Figure 3 shows three typical MS/MS spectrums of

peptides derived from low-density lipoprotein receptor-rela-

ted protein 2 precursor. The three predigested peptides were

separated into fraction 1, 5 and 7 by OGE. The spectra

represent the amino acid sequences of tryptic peptides,

(R)IDMVNLDGSYR(V), (R)GIAVDPTVGYLFFSDWESLS-

GEPK(L) and (R)HLCHCEEGYILER(G).

Figure 5. MW and pI distribution of the identified proteins. Largeproportions of proteins with extreme MW (o20 000 or 4100 000)or extreme pI value (especially basic proteins) were identified byusing peptide OGE.

Figure 6. Efficiency of peptide ligand libraries treatment ofurinary exosomes. Histograms show the number of MS/MSqueries, identified peptides and identified proteins in nanoHPLC-chip-MS/MS analysis of either nontreated sample or urinaryexosomes treated with peptide ligand libraries beads.



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Figure 4 shows the quantity of proteins identified with

different numbers of matching peptides in 12 wells sepa-

rately. Most of the identified peptides and proteins lied in

the fraction 1 and 5–8. The presence of large amount of

identified peptides in fraction 1 showed that there were

many acidic peptides present in the digested sample and

most of the identified peptides were neutral-based on the

presence of large amount of peptides in fraction 5–8.

Proteins with extreme MW (o20 000 or 4100 000) or

extreme pI value (especially basic proteins) are difficult to be

well mapped on a traditional 2-D-gel. However, by using

peptide OGE, proteins characterized by subsequent MS/MS

analysis were based on the MW and pI of the predigested

peptides instead of their own MW and pI, which led to the

identification of many of such proteins in our work. From all

the 512 unique proteins identified in 12 fractions, the pI range

was between 3.56 and 12.17, while 19% (95 out of 512) were

highly basic proteins with pI greater than 9, and of which, only

eight proteins were identified in a previous urinary proteomic

research [28]. Meanwhile, 27 and 10% were proteins with MW

4100 000 and o20 000 relatively. The percentage of proteins

with extreme MW and pI value is illustrated in Fig. 5. From

this point of view, peptide OGE coupled with MS/MS is a good

complement to traditional 2-D gel separation method, which

also represents an in-depth proteomic profiling of human

urinary exosomes proteome [29].

Our results demonstrated that the analysis of predi-

gested peptides by OGE fractionation provided abundant

information of the urinary exosomes proteome, notably

because OGE fractionation reduced the sample complexity,

which was advantageous for LC-MS/MS analysis.

3.4 Protein identification by nanoHPLC-chip-MS/MS

analysis

After peptide OGE, the peptides were present in the liquid

phase and can be recovered conveniently from the wells for

further processing. To go further in the characterization of

urinary exosomes proteome, each 20 mL of CPLL-treated

peptides of 12 fractions were analyzed by nanoHPLC-chip-

MS/MS. HPLC-chip is a microfluidic chip-based device that

can carry out nanoflow HPLC. Compared with conventional

column-based nanoflow HPLC, HPLC-chip offers unparal-

leled ease of use, greater reliability and robustness and higher

sensitivity. The MS/MS data were then automatically searched

against UniProtKB/Swiss-Prot protein database by Spectrum

Mill Proteomics Workbench Rev A.03.03.078 software. More-

over, to evaluate whether the peptide library treatment

provided a real advantage in terms of number of proteins

identified by nano-HPLC-chip-MS/MS, the analysis on the

crude urinary exosomes sample before any treatment was also

performed. A total of 2949 nonredundant peptides in CPLL-

treated urinary exosomes were determined, leading to the

identification of 512 nonredundant proteins, which represent

an in-depth characterization of this material compared to the

nontreated sample as illustrated in Fig. 6. Of the identified

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512 proteins, there were 174 proteins, which have at least two

nonredundant peptide identifications. Comparing the

proteins identified by the two methods (with or without CPLL

treatment), there were 188 common proteins identified by

both methods, whereas 23 and 324 proteins were found only

in the nontreated or CPLL-treated sample. Figure 2(B) shows

the distribution of the number of proteins identified by both

methods.

The identification results demonstrated the potential

application of a parallel MS/MS analysis coupled with peptide

OGE for complicated protein sample identification, especially

for proteome analysis, such as bio-fluids or tissues. Other

datasets of urinary exosomal proteins have been published in

the past. For instance, using 1-D SDS/PAGE fractionation

and nanospray LC-MS/MS, 295 unique proteins were iden-

tified [5]. Recently, after depletion of THP by adding DTT into

the sample, a proteomic analysis of urinary vesicles through a

high-sensitivity linear ion trap mass spectrometer identified

more than 1000 proteins in urinary exosomes [11]. Combined

with our result, it is suggested that urinary exosomal

proteome is unexpectedly large, and there are large propor-

tion of low-abundance proteins in urinary exosomes. Table 1

lists several proteins that may be interesting in potential

biomarker discovery of renal diseases.

Using a platform of nanoHPLC-chip-MS/MS combined

with CPLLs and peptide OGE as described in this paper, we

have been able to improve the separation and identification

of proteins in urinary exosomes. As a matter of fact, the

major reason that accounts for the relatively high total

number of protein species reported here, as compared with

the crude urinary exosomes before any treatment, rests most

probably on the application of the hexapeptide ligand library

technology. That is because it namely has the ability of

sharply cutting the high-abundance proteins while greatly

enriching the low-abundance species until full saturation of

the corresponding ligand in the library.

3.5 GO annotation

Finding functional proteome is the main aim for mapping

global expression patterns of proteins. The rich information

from this proteomic analysis provides further insight into

the biogenesis of urinary exosomes.

By submitting the IPI numbers of identified proteins to

GO Term Finder of the Lewis–Sigler Institute, it was

determined that 167 identifiers were not annotated and the

other 345 placed were categorized by ontological aspects.

Figure 7. Distribution of the GO cellularcomponent, biological process and molecularfunction aspect for 512 identified proteins inlow-abundance proteome of urinary exosomes.Assignments were made using GO Term Finderof the Lewis–Sigler Institute. The ordinate valueis the number of proteins that match a specificsubcategory of this aspect.



Proteins were analyzed and categorized based on their

cellular component, biological process and molecular func-

tion.

For the cellular component of the detected proteins

illustrated in Fig. 7, the top three categorizations fell in

intracellular compartments (243 of 345 proteins, 70.43%),

cytoplasm (191 of 345 proteins, 55.36%) and membrane

(155 of 345 proteins, 44.93%). With respect to biological

process, 70.72% of proteins were related to cellular process,

49.86% were involved in the metabolic process and 39.71%

were predicted to be involved in regulation of biological

process. The subcategory of molecular function included a

high proportion of proteins that were involved in binding

and catalytic activity.

4 Concluding remarks

Although the urinary exosomal proteome only accounts for

3% of total urinary proteins, it is unexpectedly complex and

may prove useful in biomarker discovery in the future. Our

results of analysis of low-abundance proteome of !urinary

exosomes may contribute to the understanding of

molecular composition and formation process of urinary

exosomes.

The combination of peptide fractionation by OGE and

nanoHPLC-chip-MS/MS analysis of CPLL-treated sample

allowed the idenfication of hidden proteome from urinary

exosomes, especially proteins with extreme MW or extreme

pI value. More importantly, the introduction of such a novel

way rendered possible the concentration and identification

of low-abundance protein species from complex proteomes

of bio-samples.

The authors have declared no conflict of interest.

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comprehensive analysis of low-abundance proteins in human urinary exosomes using peptide ligand...

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