June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Short Communication

Recovery of extracellular vesicles from liquid samples
using polyamine solution
1,2
1
1,3
2
2
Arizumi Kikuchi *, Azumi Naruse , Kenichi Nonaka , Motoki Mori , Miyuu Yamada ,Keiko Kano , Emi Mishiro-Sato , Kaname Tsutsumiuchi
442

Daiyukai Research Institute for Medical Science, 25 Azaicho, Ichinomiya, Aichi 491‒0113, Japan
College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai-shi, Aichi 487‒8501, Japan3
Department of Surgery, Daiyukai General Hospital, 1‒9‒9 Sakura, Ichinomiya, Aichi 491‒8551, Japan
4
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464‒8601, Japan
1

2

Abstract

Liquid biopsies mainly analyze nucleic acids and proteins in the free-state or extracellular vesicles (EVs) in

non-solid biological samples, primarily blood. Collecting and processing liquid biopsy samples is challenging due to thelarge volume of samples and reagents and the need for special equipment. In a previous study, we reported a method forenriching free nucleic acids using a polyamine solution that is eﬀective for liquid biopsy. We also investigated the reactivity of this method for EV recovery in cell culture supernatants using mass spectrometry. Samples were prepared from thecell line NCI-N87 supernatants after 48 h of culture in a serum-free medium. For comparison, samples were treated using asolution containing polyamines (PA method) or ultracentrifugation (UC method). The liquid chromatography-tandem mass
spectrometry (LC-MS/MS) using the single-pot solid-phase-enhanced sample-preparation (SP3) method revealed diﬀerences between the two methods in the total ion chromatogram of the sample. However, the results of the Gene Ontology (GO)analysis showed that both methods achieved the best enrichment in GO terms related to EV. In addition, the volcano plot
analysis revealed that proteins suggested to exist in EVs were distributed in areas consistent with both methods. These results indicated that the PA method can recover EV proteins in liquid samples, and their comprehensive analysis is possibleusing the SP3 method.
Key words: extracellular vesicles, liquid biopsy, LC-MS/MS, polyamine method, single-pot solid-phase-enhanced samplepreparation (SP3) method

Introduction

state nucleic acids and proteins in extracellular vesicles

Blood samples from patients with cancer contain circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), circulat-(EVs), including exosomes . Liquid biopsy can provide
information on the nature of cancer without requiring inva-

ing tumor cells, and microRNAs (miRNAs) derived fromsive procedures associated with conventional tissue collec-

2)

1)

3)

cancer cells . Liquid biopsy is a method for the genetic and1)cytological analysis of these samples . It analyzed free-tion .
EVs, including exosomes, contain proteins, mRNA,
miRNA, and DNA on their surface or inside; they are stable*Corresponding author

in body ﬂuids such as blood, urine, saliva, spinal ﬂuid, andArizumi Kikuchi

4)

breast milk after being secreted from cells . EVs have

Daiyukai Research Institute for Medical Science, 25 Azaicho,
Ichinomiya, Aichi 491‒0113, Japan

attracted attention as critical biological factors that convey
messages locally and remotely and are deeply involved invarious diseases and biological phenomena, including can-Tel: ＋81‒586‒533661, Fax: ＋81‒586‒533771
E-mail: akikuchi@daiyukai.or.jp

5)

cer and immune and neurological diseases . They can berecovered using centrifugation, immunoprecipitation, parti-

Received: February 13, 2024. Accepted: May 9, 2024.
Epub June 5, 2024.

cle size classiﬁcation, column adsorption, or ultracentrifu-

DOI: 10.24508/mms.2024.06.006
35

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Medical Mass Spectrometry Vol. 8 No. 1

6)gation . Each method has diﬀerent recovery rates, speciﬁci-

Polyamine method

ties, and the time and cost required for operation.

To begin with, 9.0 mL of the treated cell culture superna-

Therefore, the method selected depends on its application.
Although ultracentrifugation is considered the gold stan-tant was transferred into a 15 mL conical centrifuge tube
(Nunc, ThermoFischer Scientiﬁc, USA). Then, 1/10

7)

dard , it has limitations, such as poor recovery rates, andvolume of 5 M NaCl solution and 1/10 volume containing
0.01% Spermidine (191-13831, FUJIFILM Wako

8)

requires time and specialized equipment . Moreover, weconsidered the possibility that the PA method coprecipitates
EVs along with nucleic acids. In a previous study, we have

Chemicals, Osaka, Japan) were added to the tube. After the
mixture was allowed to stand for 10 min at room tempera-

devised a method for enriching free nucleic acids using
9)
polyamine-containing solutions .

ture, it was centrifuged at 27,210×g (13,000 rpm) for
60 min using a micro-cooled centrifuge (Model 3700,

Here, we investigated, using liquid chromatography-mass
spectrometry (LC-MS/MS), whether this method can be

KUBOTA CORPORATION, Tokyo, Japan). Then, the cellused to recover EV proteins in cell culture supernatant.

before analysis.

Materials and Methods

Mass spectrometry

culture supernatant was removed and dissolved in PBS

Cell culture

Sample preparation was performed using the single-pot
solid-phase-enhanced sample-preparation (SP3) technology.

NCI-N87 (ATCC, Manassas, VA, USA).
The cells were cultured in RPMI-1640 with L-Glutamine

The protein in the sample was adjusted to 1‒200 μg/mL,

and Phenol Red (Fuji ﬁlm Wako, Tokyo, Japan) with 10%

25 mM. After 30 min at room temperature, 1/10 volume of
500 mM 3-methyl-1-butanol was added and allowed to

For our LC-MS/MS analysis, we used a cell line, that isand dithiothreitol was added to a ﬁnal concentration of2

fetal bovine serum at 37°
C in a 5% CO incubator. When
Advanced RPMI 1640 Medium (Thermo Fisher Scientiﬁc,stand for 30 min at room temperature. Particles and SeraTM
SpeedBead Carboxylate-Modiﬁed [E3] Magnetic
Mag

Waltham, MA, USA).

Particles (Global Life Sciences Technologies Japan K.K.,

the cells reached conﬂuency, they were treated with

After 48 h of culture, the collected media were ﬁrst

Tokyo, Japan). The bead mixture (10 μL) was added to thecentrifuged at 1,000×g for 10 min at room temperature(20‒25°C) to pellet and remove cells. All the centrifugation

sample for 10 μg of protein. Then, three times the volume

steps were performed at 4°
C. Next, the supernatant wasfor 10 min at RT, and 500 μL of 80% ethanol was added,

centrifuged at 2,000×g for 20 min. The media supernatant
was ﬁltered through a 0.22-μm pore ﬁlter (HAWACH

followed by washing three times using a magnetic stand.

Scientiﬁc Co. LTD, Xian City, China). Each method waswas added. After sonication, the mixture was incubated at

performed using a ﬁltered culture medium.

37°
C for 3 h. Trypsin solution was added, and the sampleof ethanol was added, the sample was mixed at 1,000 rpm

Then, 100 μL of lysyl endopeptidase plus 50 mM Tris buﬀer

was mixed at 1,500 rpm at 37°
C. for 20 h. After mixing,

Ultracentrifugation
Treated cell culture supernatant (50 mL) was used for

20% triﬂuoroacetic acid was added to the volume 1/20, thebeads were attached to a magnetic stand, and the superna-

ultracentrifugation to collect EVs. The ultracentrifugation
was performed using an Optima L-90K ultracentrifuge

tant was collected. Then, the supernatant was desaltedusing GL-Tip SDB (GL Sciences Inc., Tokyo, Japan). The(Beckman Colter, Fullerton, CA, USA) at 150,000×g forresulting solution was vacuum dried, and resuspended in

100 min in a SW28.1 Roter Swinging Bucket rotor(Beckman Colter). After centrifugation, phosphate-buﬀered

0.1% TFA in 2% acetonitrile to extract the peptides.
The LC-MS/MS analysis was performed using a PAL

saline (PBS) was added to the top of the tube, and ultracen-

Systemtrifugation was performed again under the same conditions.

Switzerland) and Q Exactive Hybrid Quadrupole-Orbitrap

Finally, the supernatant was removed, and the collected

Mass Spectrometer (Thermo Fisher Scientiﬁc). The samples
were concentrated on a C18 trap column (5-μm particle size,

EVs were suspended in PBS.

autosampler(CTC Analytics AG,

Zwingen,

300 μm inner diameter, 5 mm length; Chemical Evaluation
and Research Institute, Tokyo, Japan) and separated on a36

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

C18 column (3-μm particle size, 100-μm inner diameter,

10)

accession ID, PXD035832) .

125 mm length; Nikkyo Technos, Tokyo, Japan) at a ﬂow

Furthermore, we used the proteins described in
6)

MISEV2018

and examined the validity of the proteinsrate of 0.5 μL/min with an injection volume of 7.5 μL. The
mobile phases were solvent A (0.5% acetic acid) and sol-detected. Among the proteins listed, we identiﬁed peptide

vent B (0.5% acetic acid in 80% acetonitrile). The elution

spectrum matches (PSM) for signiﬁcant proteins. We also

gradient for solvent B was as follows: 5% to 40% B overexamined the correlation between the two methods using

100 min, then 40% to 95% B for 1 min, holding at 95% B

this data.
We also examined the properties of the PA and UC meth-for 3 min, then back to 5% B over 1 min, and ﬁnallyre-equilibrating at 5% B for 10 min. Electrospray ionization

ods using total ion chromatogram, diagrams, and volcano

was performed in positive-ion mode.
The instrument was operated in the DDA mode. Xcalibur

plot, mainly to characterize the recovered peptides. The
volcano plot was also used to examine both methodsʼ agree-

4.1.50 (ThermoFisher Scientiﬁc) was used to record

ment rate and characteristics of the proteins detected.

peptide spectra. The full scan was acquired from 350 to1800 m/z with a resolution of 17,500, automatic gain con-

Volcano plots were created with a fold change value (log2)
greater than or equal to 1.5 and a p-value of 0.05＞. The

6trol (AGC) as 3×10 , and maximum injection time as

methodʼs relative standard deviation (RSD) was determined

60 ms. MS/MS scans were performed with a resolution ofand reproducibility was discussed. RSD was calculated by

535,000, AGC target as 1×10 , and maximum injection timeﬁnding the mean and SD of the PSMs listed in Table 1.

as 60 ms. The 10 highest intensity precursor ions wereWe performed Go Ontology (GO) analysis on cellularcomponent categories using proteins detected by both meth-isolated using the quadrupole analyzer in a window of
2.0 m/z and fragmented by higher energy collisional dissoci-ods for PSMs with a mean of 4 or higher. The analysis used
11,12).
the DAVID open-source program, v2024q1

ation (HCD) fragmentation with normalized collisionenergy (NCE) of 27%. Multiply-charged peptides were

Results

chosen for MS/MS experiments. Dynamic exclusion time
was set to 20 s.

Table 1 shows the protein data obtained by both methods,

Data analysis

using the detection data for the content described in
MISEV2018 as protein content-EV characterization, and

UniProt Homo Sapiens (TaxID＝9606) protein sequence
database downloaded on 09/24/2022 and cRAP for contam-

summarized for those with PSMs of 4 or higher. Using

inants (http://www.thegpm.org/crap/) were used. MS/MS

PSMs, a correlation coeﬃcient of R＝0.765 was obtained
for both methods, resulting in an overall positive correla-

spectra were interpreted, and peak lists were generated

tion in Fig. S1. In addition, results for all proteins listed inusing Proteome Discoverer 2.5.0.400 (ThermoFisher

MISEV2018 are represented in Table S1.

Scientiﬁc). Searches were performed using SEQUEST

Fig. 1A shows the total ion chromatograms. Although the(ThermoFisher Scientiﬁc). Search parameters were set assame samples were treated using PA and UC methods, eachfollows: enzyme selected with two maximum missing

method showed a diﬀerent proﬁle.

cleavage sites, a mass tolerance of 10 ppm for peptide

Fig. 1B show the results of the volcano plot for eachtolerance, 0.02 Da for MS/MS tolerance, ﬁxed modiﬁcation

method. Fig. 1B shows the markers CD9, CD63, CD81,
and Flotillin-1, which were considered particularly import-of carbamidomethyl (C), and variable modiﬁcation ofoxidation (M) and N-terminal acetylation. Peptide identiﬁ-

ant in EVs. The PA method identiﬁed common proteins

cations were based on signiﬁcant Xcorr values (high conﬁ-

believed to be contained in EVs near the origin. Table S2dence ﬁlter). Peptide identiﬁcation and modiﬁcation

presents the list of proteins with quantitative information

information returned from SEQUEST were ﬁltered at aused in the analysis.
Table S3 summarizes the number and percentage of pro-false discovery rate (FDR) of 1% using the Percolator nodeof Proteome Discoverer to obtain conﬁrmed peptide identi-

teins with diﬀerent reproducibility based on the RSD values

ﬁcation and modiﬁcation lists of HCD MS/MS. The
LC-MS/MS data generated in this study have been depos-of PSMs in proteomics using PA and UC methods. About

ited in jPOST repository database (project ID, JPST001807,

smaller RSD value than 30%, whereas about 20% of the

80% of the proteins detected with the PA method have a37

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Medical Mass Spectrometry Vol. 8 No. 1

Table 1.

Protein content by LC-MS/MS
PSMs

Description

Integrin alpha-2Integrin beta-3
HLA class I histocompatibility antigen, C alpha chain
Integrin alpha-6Integrin beta-1
HLA class I histocompatibility antigen, A alpha chain
HLA class I histocompatibility antigen, B alpha chain
Integrin beta-4 OS＝Homo sapiens
Syndecan-4 OS＝Homo sapiens
CD9 antigen OS＝Homo sapiens
Receptor tyrosine-protein kinase erbB-2
Charged multivesicular body protein 1b
Annexin A7 OS＝Homo sapiens
ADP-ribosylation factor 6
Flotillin-2
EH domain-containing protein 2
Transforming protein RhoA
Flotillin-1
Annexin A13
Charged multivesicular body protein 2b
Arrestin domain-containing protein 1
Charged multivesicular body protein 1a
EH domain-containing protein 3
Vacuolar protein sorting-associated protein 4A
Charged multivesicular body protein 3
Annexin A4
Tumor susceptibility gene 101 protein
Charged multivesicular body protein 4b
Vacuolar protein sorting-associated protein 4B
Charged multivesicular body protein 4c
Annexin A3
EH domain-containing protein 4
Annexin A1
Programmed cell death 6-interacting protein
Charged multivesicular body protein 2a
EH domain-containing protein 1
Putative annexin A2-like protein
Annexin A2
Syntenin-1
Annexin A11 OS＝Homo sapiens
Glyceraldehyde-3-phosphate dehydrogenase
Actin, cytoplasmic 1
Tubulin beta-3 chain
Tubulin beta-8 chain
Tubulin beta-2B chain
Tubulin alpha-4A chain
Tubulin alpha-3E chain
Tubulin alpha-3D chain
Tubulin beta chain
Tubulin alpha-1A chain
Tubulin alpha-1B chain
Tubulin alpha-1C chain
Tubulin beta-4B chain
Tubulin beta-2A chain
Heat shock protein HSP 90-beta
Heat shock cognate 71 kDa protein
Albumin

Gene Symbol

ITGA2
ITGB3
HLA-C
ITGA6
ITGB1
HLA-A
HLA-B
ITGB4
SDC4
CD9
ERBB2
CHMP1B
ANXA7
ARF6
FLOT2
EHD2
RHOA
FLOT1
ANXA13
CHMP2B
ARRDC1
CHMP1A
EHD3
VPS4A
CHMP3
ANXA4
TSG101
CHMP4B
VPS4B
CHMP4C
ANXA3
EHD4
ANXA1
PDCD6IPCHMP2AEHD1
ANXA2P2ANXA2SDCBPANXA11
GAPDH
ACTB
TUBB3
TUBB8
TUBB2B
TUBA4A
TUBA3ETUBA3DTUBB
TUBA1A
TUBA1B
TUBA1CTUBB4B
TUBB2A
HSP90AB1
HSPA8
ALB

(a)

(b)

Mean

S.D.

Mean

S.D.

4.0
6.7
9.0
10.3
13.7
16.3
16.3
18.7
45.3
6.7
22.0
4.0
1.3
1.7
1.7
3.7
4.0
4.0
5.0
5.7
5.7
7.3
8.0
8.0
8.3
9.7
10.0
12.0
12.7
12.7
16.3
18.0
21.3
23.7
26
26.7
50.3
67.7
5.0
11.0
4.0
61.0
2.5
2.7
2.7
3.3
4.0
4.0
4.3
4.7
4.7
4.7
6.0
2.7
32.0
39.3
67.7

2.0
1.2
1.7
0.6
1.5
1.2
1.5
4.9
5.7
1.2
1.7
1.0
0.6
1.2
1.2
1.2
0.0
1.0
1.0
1.5
1.5
1.5
0.0
3.0
3.8
1.5
1.7
1.7
4.0
1.5
0.6
3.0
1.2
1.5
2.6
3.2
3.2
3.1
1.0
2.0
0.0
2.6
2.1
1.5
1.2
1.2
1.0
1.0
0.6
1.5
1.5
1.5
1.0
1.2
5.3
4.7
18.5

3.7
4.5
8.7
9.0
12.0
15.3
18.7
15.0
5.5
5.0
24.3
6.3
4.0
4.5
6.5
5.5
6.7
7.0
13.5
5.0
7.3
9.3
8.3
12.5
12.7
14.3
13.3
19.7
14.7
17.3
12.3
17.0
18.0
34.7
45.7
23.7
42
59.3
10.0
12.0
4.7
49.0
4.0
4.5
7.5
5.0
5.0
5.5
9.0
8.5
9.5
9.5
6.3
8.0
22.0
43.0
12.0

2.3
0.7
6.1
8.0
5.3
11.9
11.0
12.1
2.1
4.0
16.0
4.0
1.4
0.7
0.7
3.5
3.8
5.3
4.9
4.0
4.7
6.4
6.7
4.9
9.3
10.7
9.3
11.0
11.1
8.1
6.4
14.1
8.5
23.2
28.7
22.1
18.1
25
4.2
5.3
4.0
32.8
4.2
4.9
6.4
6.1
5.7
6.4
5.7
10.6
12.0
12.0
6.1
7.1
14.2
27.1
12.2

(a) PA method and (B) UC method. The analysis was performed with N＝3, and the list was made up of PSMs obtained by any of the methods with a mean of 4 or higher (PSMs, peptide spectrum matches; SD, standard deviation).

38

June 2024

Fig. 1.

Medical Mass Spectrometry Vol. 8 No. 1

Comparison of both methods using total ion chromatogram and volcano plot.
A. Total ion chromatogram for each method. (a) PA method, (b) UC method. B. Volcano plot for each method. Marked for proteinsin EVs at NCI-N87. Created using abundance data obtained by LC-MS/MS. The x-axis indicates log2 fold change for each gene,and y-axis indicates -log10 p-value. Red dots indicate proteins with p-values ＜0.05, and green dots indicate proteins with p-values≥ 0.05. The signiﬁcance of diﬀerences was evaluated by t-test, with p-value ＜0.05 being signiﬁcant.

proteins with the UC method.

capture tumor dynamics owing to the heterogeneous

In the GO analysis, EV markers, including exosomes,

distribution of tumor cells imposes challenges to cancer

dominated the highest annotation cluster with enrichment.

diagnosis . Meanwhile, liquid biopsy is a promising

The enrichment scores for the PA and UC methods were

method to address these challenges. In addition, liquidbiopsy results have recently been used to administer com-

14)

206.6 and 233.6, respectively (Table S4 A, B) .

panion diagnostic agents.

Furthermore, information regarding cellular components

We compared the EVs obtained by PA and UC methods
using the LC-MS/MS. Together with the UC method,

obtained in this analysis is described in Fig. S2.

Discussion

Cancer is the leading cause of death worldwide; there-

LC-MS/MS detected proteins that indicate EVs according
to the categories in the MISEV2018. The total ion chro-fore, early cancer detection and appropriate treatment are

matograms obtained using the PA method diﬀered from13)

crucial . Usually, cancer is diagnosed by tissue biopsy;

those obtained using the UC method. In the analysis withhowever, the invasive sampling and inability to accurately

the volcano plot, the common markers that were supposed39

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Medical Mass Spectrometry Vol. 8 No. 1

to be present in the EV appeared relatively close to the ori-process.

gin. However, their distribution showed diﬀerent patterns.

Liquid biopsy has been reported in urine, saliva, cerebro-

Although the UC method is the standard method for EV
recovery, it simultaneously recovers impurities such as ves-

spinal ﬂuid, and stool other than blood , and is expected to

icles and proteins other than EVs, resulting in lower purity

proteins in the samples used in liquid biopsies are often

22)

be used in actual clinical practice. Free nucleic acids and

8)

23)

of EVs . These factors may have inﬂuenced the diﬀerences

present at low concentrations

and require a large sample

observed between the two methods in the present study. In
addition, EVs contain exosomes, microvesicles, and apop-

volume. In addition, large amounts of reagents and special

totic bodies, and the reactivity of each substance in eachcan be enriched, it is possible to use ordinary processing

equipment are required. However, because the PA method15)

method , including PA method, can signiﬁcantly aﬀect thereagents to handle enriched samples.

results.

We have demonstrated the presence of protein markers in

The proteins detected by both methods are positively
correlated and show a similar trend in detecting EV-related

the characterization shown in MISEV2018 using the PA

markers. Furthermore, we believe the PA method is lessin EVs were recovered in this study. Furthermore, we
believe that a comparative study of this point using meth-method; however, it is unclear to what extent the exosomes

susceptible to contamination by dead cells. The results of
the GO analysis also suggest that this is an eﬃcient methodof recovering EV-related substances. Although the PA

ods other than the UC method should be conducted. There-

method has shown recovery of proteins in EVs, its charac-

analytical methods.

fore, we are currently examining this point using various
The cargo of tumor-derived EVs is consistent with theteristics will need to be clariﬁed through further studies.

24)

genetic content of parent tumor cells . Therefore, thePolyamines are involved in cell growth and survival,
16)

autophagy , and the onset and progression of neurological17)
disease . Additionally, a link between polyamine meta-cargo contained in the EVs has attracted attention as a new
25)

biomarker for cancer diagnosis and prognosis prediction .

18)

bolic pathways and cancer has been reported . Polyamines

Moreover, EVs are stable in body ﬂuids, and the cargo ininclude streptomycin, spermidine, and spermine, which
exist in cells at a level of several millimolar concentra-

the EVs is protected from degradation

are of interest as excellent biological components for liquid

19)
tions . Moreover, polyamines are polycationic mole-biopsy26,27)

20)

1,28). Therefore, EVs. We are currently conducting various studies

cules , which is the characteristic the PA method focuses

focusing on its application in liquid biopsy. We believe thaton.

the PA method can potentially demonstrate a broad utility

This method can recover cell-free nucleic acids and EVs
9)using polyamine-containing solutions , which we believe isby comparing its performance with that of other methods

due to the formation of ion complexes between polyamines

Mass spectrometry is being implemented in various ways

and these substances with negative charges on the surface.
Furthermore, under high salt concentration conditions, sub-in the medical ﬁeld because of its ability for comprehensive

for EV recovery.

analysis and its excellent sensitivity and speciﬁcity. We
believe that this method will be an eﬀective method in clin-stances with small particle sizes and low charge density do
21)

not lead to the formation of coacervate , which may also

ical testing of liquid biopsy samples.

aﬀect not recovering a variety of free proteins.

Conclusions

The PA method does not require special equipment, isrelatively easy to operate, and is low-cost. In addition, a
pretreatment is possible when further downstream puriﬁca-

EV analysis using LC-MS/MS with the SP3 method was
also feasible. The LC-MS/MS results showed that the PAtion or nucleic acid extraction is performed on samples col-method can detect peptides with EV properties.

lected using the PA method. Regarding the reproducibility

Conﬂict of Interestof the PA method, approximately 80% of the proteinsdetected in this study showed RSD within 30%. Maintain-

The authors declare no competing interests.

ing and improving reproducibility is an essential issue in

Additional Information

analysis. We believe that attention should be paid not only

The results of this study are organized in the text or into technical proﬁciency but also to further simplifying the40

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Medical Mass Spectrometry Vol. 8 No. 1

GB, et al: Single-pot, solid-phase-enhanced sample prepa-

supplementary materials.

ration for proteomics experiments. Nat Protoc 14: 68‒85,

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