June 2023

Medical Mass Spectrometry Vol. 7 No. 1

Research Paper

Detection of highly abundant small molecules in the stratum corneum of
healthy young women using desorption electrosprayionization-mass spectrometry imaging
1

1

2

2

2

2

A.S.M. Waliullah , Takumi Sakamoto , Tomoya Uchiyama , Eriko Itai , Eiji Naru , Ken Inomata ,
1

1

1

1,3

1

Md. Al Mamun , Md. Mahmudul Hasan , Reimu Fukui , Tomohito Sato , Yutaka Takahashi ,
4

1,3

1,3,5

Tetsuya Honda , Tomoaki Kahyo , Mitsutoshi Setou*

1

Department of Cellular & Molecular Anatomy, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431‒3192, Japan
2
KOSÉ Corporation Research Laboratories, 48‒18 Sakae-cho, Kita-ku, Tokyo 114‒0005, Japan
3
International Mass Imaging Center, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431‒3192, Japan
4
Department of Dermatology, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431‒3192, Japan
5
Department of Systems Molecular Anatomy, Institute for Medical Photonics Research, Preeminent Medical Photonics Education &Research Center, 1‒20‒1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431‒3192, Japan
Abstract

Aging reﬂects skin appearance drastically, which reduces skin juvenescence. However, the small molecules

underlying skin juvenescence have not been well studied. We aimed to explore the molecules potentially responsible foryoung-looking skin. Eleven healthy women aged 24‒59 years were recruited and divided into young and middle-aged
groups. Multiple layers of the stratum corneum from the cheek area were taken by tape-stripping, followed by desorption
electrospray ionization mass spectrometry imaging (DESI-MSI). Overall, ﬁve molecules (m/z 284.33, 340.39, 488.39,
628.37, and 863.65) were highly abundant in young subjects. Among them, m/z 284.33 and 340.39 were dominantly
detected in each layer of all young subjects. Interestingly, m/z 488.39 and 628.37 were prominent in young subject 4. All ofthese molecules were gradually decreased in the successive layers of the stratum corneum in subjects 3 and 4 of the younggroup. These molecules could be endogenous, co-related with youthful skin, or retained from topical cosmetics. Extensive
research is indispensable to characterize them and ﬁnd the relationship between these moleculeʼs retention capacity in thestratum corneum with diﬀerent skin parameters. Our ﬁndings provide a novel perspective on young skin that could be
advantageous in future cosmetic formulations to improve skin juvenescence.
Key words: stratum corneum, DESI-MSI, molecules, skin juvenescence

Introduction*Corresponding author

Skin is the most visible external attribute of the human

Mitsutoshi Setou

1)

2,3)

being . It is one of the critical indicators of aging. The
aging process signiﬁcantly impacts the skinʼs young appear-

Department of Cellular & Molecular Anatomy, Hamamatsu
University School of Medicine, 1‒20‒1 Handayama, Higashi-

4,5)

ance

ku, Hamamatsu, Shizuoka 431‒3192, Japan
Tel: ＋81‒53‒435‒2086, Fax: ＋81‒53‒435‒2468. The appearance of the skin is highly correlated tothe stratum corneum, which is the outer layer of the skin.
6)

Stratum corneum, in turn, consists of multiple layers in it .
Improved skin texture and blood ﬂow, prevention of pig-

E-mail: setou@hama-med.ac.jp

Received: July 19, 2022. Accepted: August 29, 2022.
Epub October 11, 2022.

mented spot formation, and increased moisture in the stra-

DOI: 10.24508/mms.2023.06.001

tum corneum are the external parameters for skin to have26June 2023

Medical Mass Spectrometry Vol. 7 No. 1

7)
beautiful, young-looking skin . Keratin is a major compo-

Hamamatsu University School of Medicine (Code: 19-123).

nent of stratum corneum and constitute more than 85% ofStudy subjects were provided with suﬃcient informationand signed informed consent forms before collecting tape-8)

the total proteins in it . Keratin and associated proteins are
9,10). However, until now
very few studies on the other small molecules in the stra-

stripped stratum corneum samples. Each subjectʼs stratum

tum corneum underlying skin juvenescence have been

numerical subject number.

deformed over the aging process

corneum sample was anonymized by assigning a uniquedone. In this connection, we aimed to discover the potential

Study subjects and tape stripped stratum corneum
samples collection

small molecules in the stratum corneum that might beresponsible for skin juvenescence. While it is feasible toinvestigate human skin that has been surgically resected

In this study, 11 healthy Japanese female volunteers par-during some diseases, however in healthy individuals, skinticipated. Only individuals who participated in this testing

11)

sampling is desired to be noninvasive . Several studies
have recently been employed in normal conditions to analyze skin

7,12,13)schedule could consent to the stratum corneum sampling
and other test items, and not suﬀering from skin diseases. Recently, several techniques have been

like eczema or other pathological conditions were selected.
Subjects who recently received special facial care or medi-

employed to analyze skin conditions by imaging the skin7,14‒16). Apart from these,
based on its external appearance
mass spectrometry based studies are a more advanced tech-cation that may aﬀect the sample collection site (face) were17,18)

chosen as subjects to eliminate sexual diﬀerences between

excluded from this study. In addition, only women were.
Among the mass spectrometric methods, the mass spec-nique due to providing molecular information as wellmen and women. Six subjects were categorized as young,aged between 20‒30s. (subject number 1 to 6 in the young

trometry imaging (MSI) analysis adds a new dimension to19‒23). Curthe biomolecular distribution analysis in the skingroup). The other ﬁve subjects were between the ages of40‒60s and categorized as middle-aged (subject number 7

rently, multiple MSI modalities are in use, like matrix-assisted laser desorption/ionization (MALDI)-MSI, second-

to 11 in the middle-aged group) (Table 1). As a samplingary-ion mass spectrometry (SIMS) imaging, and DESIMSI. Compared to other MSI modalities, DESI-MSI is aarea, we selected the cheek area of the face. Redness, pores,

rapidly in-situ molecular imaging technique because it does
not require matrix coating on the sample, unlike MAL-

transparency was scored by visual evaluation. The redness,pores and spots scores were scored on a 5-point scale from

DI-MSI. Moreover, DESI-MSI is used to detect compounds

0 to 2 with 0.5 increments. The lower score of redness,pores, and spots indicates better skin condition and is con-and spots were scored by photo evaluation, while skinand visualize their spatial distribution in the sample in
11,24‒27)

ambient conditions. For straightforward, rapidness,sidered to have a youthful appearance of the skin. The skintransparency score was scored on a 9-point scale from 0 to

and direct surface analysis capacity in ambient condition, in
this study, we employed DESI-MSI to detect small mole-

4 with 0.5 increments. Lower scores indicated higher skintransparency. The skin with more increased skin transpar-

cules of stratum corneum, which are highly abundant in28)
young-aged skin .

ency is typically considered to have a youthful appearanceof the skin. These redness, pores, spots and skin transpar-

Materials and Methods

ency score evaluation was constructed based on the “Skin29)
Aging Atlas” exclusive to Asian type . Before stratum cor-

Reagent and chemicals
Acetonitrile, methanol, formic acid, ultrapure water, andneum sample collection, each subjectʼs face was thoroughly

isopropanol were purchased from FUJIFILM Wako pure

washed with the same (commercial) mild cleanser (Table

chemical industries (Osaka, Japan). Leucine enkephalin

S1) using tap water. After face washing, the subjects wereallowed to acclimate in a constant temperature and humidi-

was purchased from Waters Corporation (Milford, MA,
USA). Sodium formate was obtained from Sigma-Aldrich
Co. LLC (St. Louis, MO, USA).

ty-maintained room (room temperature 20±2°
C, humidity
50±5%) for 15 minutes at least. The stratum corneum sam-

Ethicsples (horny layers of the epidermis) were then collected by
2
1 cm tape-stripping by cellophane tape (CELLOTAPE™,
CT-18, NICHIBAN Co., Ltd., Tokyo, Japan) from the

The study was approved by the ethics committee of the27June 2023

Medical Mass Spectrometry Vol. 7 No. 1

Table 1.
Subject
group
Youngaged

Middleaged

Demographic data of the donors of tape stripped stratum corneum sample

Subject ID

Age(years)Redness(0 to 2scale)

Pores(0 to 2scale)

Spots(0 to 2scale)

Skin transparency (0to 4 scale)

Skin conditions

1

27

1.5

1.5

0.5

3.5

Redness, many bumps due to acne scars, normal skin tone

2

30

1.5

1.5

0.5

3.5

Many bumps due to worsening acne, normal skin tone

3

31

0.5

0.5

0.5

3.5

Many bumps due to acne scars, normal skin tone

4

25

0.5

0.5

0

0

5

28

0

0

0.25

1

Brighter skin tone, almost no pigmentation, less unevenskin surface
Lighter skin tone with unevenness due to ﬁne pigmentation, less uneven skin surface

6

24

0

0

0

1

Lighter skin tone, few bumps on the skin surface, but
bumps near the nose

7

59

0

0

2

3.5

Uneven skin tone due to hyperpigmentation, skin surface
irregularity is not worse than that of the same age group,skin tone is darker

8

55

0

2

2

3.5

Many uneven colors due to pigmentation, uneven skinsurface due to ﬂowing texture, dark skin tone

9

45

0.5

0.5

0.5

2

Less unevenness in skin tone due to pigmentation than thesame age group, skin surface irregularity about the sameas the same age group, lighter skin tone

10

41

0.5

0.5

0.5

2

11

43

0

0

0.25

2

Uneven skin tone due to pigmentation, less uneven skinsurface than same age group, lighter skin tone
Lighter skin tone with very little unevenness due to pigmentation

cheek four times from the same spot. Although the thickness of stripped stratum corneum samples was not mea-

scanned with a scan rate and pixel size of 200 μm/sec and200 μm×200 μm, respectively. A solvent pump (ACQUITYUPLC Binary Solvent Manager, Waters Corporation, Mil-

sured, we tried collecting stratum corneum samples fromeach subject with a similar method to minimize theremoved stratum corneum thickness variation between sub-ford, MA, USA) was used to supply the solvent (98 : 2

jects. The ﬁrst stripping refers to the stratum corneumʼs out-

resolving power and mass window were set at 20000 andermost layer, whereas subsequent strippings refer to thestratum corneumʼs inner layers. In all cases, the tape-0.02 Da, respectively. The DESI source conditions were

stripped samples were preserved at −80°
C until mass spec-

nitrogen gas pressure of 0.4 MPa, and (iii) inlet temperaturetrometric analysis.

of 120°C. Analyzer mode was set as “sensitivity.” Massmethanol/water, v/v) at a ﬂow rate of 2μL/min. Mass

optimized as follows: (i) capillary voltage of 3.0 kV, (ii)spectra were collected in a mass range of m/z 100 to 1000.

DESI-MSI analysis

The sodium formate solution (500μM) in isopropanol:

For DESI-MSI of the stratum corneum sample, tape-water (90 : 10, v/v) was used to calibrate the DESI mass

stripped samples were mounted on regular glass slides(Matsunami Glass Ind., Ltd., Kishiwada, Japan). A dou-

spectra externally, and the detector setup was performed

ble-sided tape (Conductive tape assy, 241-08728-92, Shi-correction option was used for mass accuracy corrections

madzu Corporation, Kyoto, Japan) was attached to the glass
slide before attaching the non-adhesive side of the strippedusing m/z 309.2036 (Na adduct of diisopropyl sebacate, atypical background peak in mass spectrometry).

tape. The analysis was conducted with a quadrupole timeof-ﬂight (Q-TOF) mass spectrometer (Xevo G2-XS Q-TOF,

Data analysis

using leucine enkephalin solution (500 μM). The lock mass＋

Waters Corporation, Milford, MA, USA) in positive ioniza-

The data acquisition and processing were made using the
MassLynx (Version 4.1; Waters Corporation, Milford, MA,

tion mode. The selected areas on the glass slide were28

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Medical Mass Spectrometry Vol. 7 No. 1

USA) program for DESI-MSI experiments. Raw data
derived from the DESI-MSI experiment was imported into

MSI ion image between groups (Fig. S3). Thus, we have

HDImaging software (Version 1.4; Waters Corporation,
Milford, MA, USA). The candidate selection was per-candidates. Background peaks were excluded considering
the moleculeʼs distribution in the negative control tape sam-formed from the list of 300 most abundant m/zs generated
in HDImaging software and manually compared the DESI-

ple. This negative control tape sample was the only tapesample attached to the glass slide, hence free from the stra-Fig. 1.

considered the average intensity between groups in picking

Average DESI-MSI mass spectrum from tape stripped stratum corneum.

(a) full positive-ion mass spectrum ranged m/z 100‒1000. (b) Expanded view of masses ranged 200‒400 showing detection of m/z
284.33, and m/z 340.39. (c) Expanded view of masses ranged m/z 400‒600 showing detection of m/z 488.39, and (d) Expanded
view of masses ranged m/z 600‒900 showing detection of m/z 628.37, and m/z 863.65.

29

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Fig. 2.

Medical Mass Spectrometry Vol. 7 No. 1

Prominent distributions of highly abundant molecules in the tape-stripped stratum corneum samples from young
and middle-aged subjects.

(a) Stratum corneum sample orientation in glass slide. (b) DESI-MSI ion images of m/z 284.33, (c) average intensity comparisonof m/z 284.33 between young and middle-aged subjects. (d) DESI-MSI ion images of m/z 340.39, (e) average intensity comparisonof m/z 340.39 between young and middle-aged subjects. (f) DESI-MSI ion images of m/z 488.39, (g) average intensity comparisonof m/z 488.39 between young and middle-aged subjects. (h) DESI-MSI ion images of m/z 628.37, (i) average intensity comparisonof m/z 628.37 between young and middle-aged subjects. (j) DESI-MSI ion images of m/z 863.65, (k) average intensity comparisonof m/z 863.65 between young and middle-aged subjects. (l) DESI-MSI ion images of m/z 414.43, a compound showing similar
distribution in young and middle-aged subjects, (m) average intensity comparison of m/z 414.43 between young and middle-aged
subjects. All values are presented as mean±SD and p values were calculated between groups by Studentʼs unpaired two-tailed t-test.

30

June 2023

Fig. 3.

Medical Mass Spectrometry Vol. 7 No. 1

Average intensity of m/z 488.39, m/z 628.37, and m/z 414.43 in each time of tape stripped stratum corneum samples.
(a) Average intensity of m/z 488.39 in ﬁrst, (b) second, and (c) third stripped stratum corneum. Average intensity of m/z 628.37
in ﬁrst (d), second (e), and third (f) stripped stratum corneum. Average intensity of m/z 414.43 in ﬁrst (g), second (h), and third(i) stripped stratum corneum as control. All values are presented as mean±SD.

tum corneum sample. Region of interest (ROIs) was manually drawn on each sample area to calculate average inten-(m/z 284.33, m/z 340.39, m/z 488.39, m/z 628.37, and m/z
863.65) are highly abundant in the stratum corneum of theyoung-aged subject compared to the middle-aged subjects

sity using HDImaging software. Average signal intensities
of individual pixels of the ROIs (Fig. S1b) were then com-

(Fig. 1b‒d and Fig. 2b‒k). Among them, m/z 284.33 waspared for relative abundance.

122%, m/z 340.39 was 196%, m/z 488.39 was 345%, m/z
628.37 was 265%, and m/z 863.65 was 137%, highly abun-

Results

dant in average intensity in young-aged subjects compared
to middle-aged subjects. m/z 414.43 is a molecule shown

DESI-MSI detected highly abundant molecules inyoung-aged subjects

here as a control, which was not much diﬀerent in distribution among the young and middle-aged groups (only 7%

In positive ion mode, we analyzed the DESI ion distribution of tape-stripped stratum corneum samples. We

higher in the young group) (Fig. 2l, m). Among all detected

explored the most abundant 300 DESI-MSI peaks between

candidate molecules, m/z 284.33 and m/z 340.39 werem/z 100 to m/z 1000 and, on average, found ﬁve molecules

prominently detected in each layer of all young subjects
31June 2023

Fig. 4.

Medical Mass Spectrometry Vol. 7 No. 1

Stratum corneumʼs layer-wise average intensity of the candidate molecules in subject 3 and subject 4 from the younggroup.
(a) Average intensity of m/z 284.33, (b) m/z 340.39, (c) m/z 488.39, (d) m/z 628.37, (e) m/z 863.65, and (f) m/z 414.43 (as control)in young subject 3. Average intensity of (g) m/z 284.33, (h) m/z 340.39, (i) m/z 488.39, (j) m/z 628.37, (k) m/z 863.65, and (l) m/z
414.43 (as control) in young subject 4.

(Fig. 2b, d and Fig. S2a‒h).
Young-aged subject 4 showed the most prominent distribu-

explore stratum corneum molecular abundance, our study
focused on the cheek area of the face as a representative
part of the body to study age-dependent molecular abun-tion of m/z 488.39 and m/z 628.37

22)Although the candidate molecules were highly abundant

dance . Among the candidate molecules, m/z 284.33 andon average in the young subjects, we discovered the mostprominent distribution of m/z 488.39 (Fig. 3a‒c) and m/z

m/z 340.39 prominently exist in all stratum corneum of all
young subjects (Fig. 2b, d and Fig. S2a‒h). Our ﬁnding sur-

628.37 (Fig. 3d‒f) in subject number 4 from the youngaged panel. By analyzing the average intensity of each stra-mises that these molecules might be highly related to skinjuvenescence. However, our study is limited to positive-ion

tum corneum layer, we also discovered that the intensity

mode-based detection of the potential candidate molecules
highly relevant for young-aged skin. The addition of nega-

was gradually decreasing in the deeper layers of the stratum
corneum (Fig. 2f, h).

tive ion mode, molecular identiﬁcation by comprehensive
analysis using liquid chromatography-mass spectrometry,

Candidate molecules were gradually decreased indeeper layers of the stratum corneum of young subjects 3 and 4

liquid chromatography with tandem mass spectrometry, andfurther functional studies are required to determine these32)
moleculeʼs relationship with young-aged skin . Interestingly subject 4 of the young-aged subjects showed a most

Although, on average, all candidate molecules are rich inyoung-aged subjects, interestingly, they are most abundant

prominent distribution of m/z 488.39 and m/z 628.37 in her
stratum corneum (Fig. 3a‒f). This most prominent abun-in the ﬁrst stripped stratum corneum of the young subject 3(Fig. 4a‒e) and 4 (Fig. 4f‒j). These moleculeʼs abundance isdance reﬂects the unique properties of this young subject,gradually decreased in the subsequent strippings. That

who has a very smooth skin surface with no pigmentationrefers to the gradual decrease of the candidate molecules inthe deeper layers of the stratum corneum of the young-aged(Table 1). In the future, it would be beneﬁcial to deepen thesubjects 3 and 4.

between its skin parameters and the retention capacity of

Discussion

those molecules. Also, we observed a gradual decrease of
the candidate molecules only (Fig. S4 a‒f) in the subse-

research on this subject to ﬁnd out the exact relationshipIn this investigation, we could detect abundant small

quent tape strippings of subjects 3 and 4. We surmised thatsome specialty, like reduced cell numbers in the deeper lay-molecules from the stratum corneum of young, healthy
individuals using the robust measurement ability of DESI30,31)

MSI

ers of the stratum corneum of these two subjects, might22)
reﬂect such distributions . In the future, it would be infor-. Unlike earlier studies that employed DESI-MSI to32

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

mative to study the exact relationship between these sub-5) Bissett DL, Oblong JE, Berge CA: Niacinamide: A B vita-

jectʼs stratum corneum and the candidate moleculeʼs gradualmin that improves aging facial skin appearance. Dermatol

decrease.

Surg 31: 860‒865, 2005.

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compositional diﬀerence in the skin

We successfully detected higher distribution of some small
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moting skin juvenescence. In the future, our ﬁndings may9) Rawlings AV: The stratum corneum and aging, in Farage

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Acknowledgements

Aging-associated alterations in epidermal function anding public utilization of advanced research infrastructure

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This work was supported by MEXT project for promot-

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