BIG DATA: MASS SPECTRAL STUDIES ON

THE HUMAN SKIN SURFACE FOR MALARIA

VECTOR CONTROL APPLICATIONS

Madelien Wooding

Yvette Naudé

Egmont Rohwer

Department of Chemistry1

September 2019

BACKGROUND

2

NH3

CO2

CO2

NH2

OH

HO

OH

O

HO

O

To chemically profile the human skin surface

with the aim of identifying compounds

which can be used in integrated malaria

vector control strategies

Chemical profile of the human skin surface

Volatolome

Contact chemical compounds

GCxGC-TOFMSVOCs

Semi-volatile organic compounds

PDMS loop

(ankle vs wrist)

TD directly in GC inlet liner

Control

Method blank

UPLC-HRMS

Semi-volatile organic compounds

Non-volatile (larger molecules)

PDMS loop

(ankle vs wrist)

Back extraction

Control

Method blank

PROJECT OVERVIEW

3

IMSAdd unique feature for

compound identification

SKIN SAMPLING METHODS

Cotton pads – back extracted [1]

Dynamic headspace adsorption onto various polymers [1]

Solid phase microextraction (SPME) [1]

Glass beads – back extracted [2]

Silicone sheets – thermal desorption [3]

Worn nylon stockings & T-shirts [4]

Body chamber [5]

Air entrainment [6]

PDMS anklets and bracelets – Alex Roodt

41. Dormont, L., J.-M. Bessière, and A. Cohuet, Human skin volatiles: a review. Journal of chemical ecology, 2013. 39(5): p. 569-578.

2. Qiu YT, Smallegange RC, Hoppe S, van Loon JJ a, Bakker E-J, Takken W. Behavioural and electrophysiological responses of the malaria mosquito Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) to human skin emanations. Med Vet Entomol. 2004;18(4):429–38. Riazanskaia

S, Blackburn G, Harker M, Taylor D, Thomas CLP. The analytical utility of thermally desorbed polydimethylsilicone membranes for in-vivo sampling of volatile organic compounds in and on human skin. Analyst . 2008;133(8):1020.

4, Takken, W. and B.G. Knols, Odor-mediated behavior of Afrotropical malaria mosquitoes. Annual review of entomology, 1999. 44(1): p. 131-157.5. Mochalski, P., et al., Monitoring of selected skin-and breath-borne volatile organic compounds emitted from the human body using gas chromatography ion mobility spectrometry (GC-IMS). Journal of Chromatography B, 2018. 1076: p. 29-34.

6. Robinson, A., et al., Plasmodium-associated changes in human odor attract mosquitoes. Proceedings of the National Academy of Sciences, 2018. 115(18): p. E4209-E4218.

Air entrainment [6]

EXPERIMENTAL

Single subject, multiple day (n=5) sampling events

Mosquito biting preference [7]

5 day study

Anklets & Bracelets

X 3 biological repeats

5

7, Braack, L., et al., Biting behaviour of African malaria vectors: 1. where do the main vector species bite on the human body? Parasites & vectors, 2015. 8(1): p. 76.

S i

C H 3

H 3 C

C H 3

O S i O

C H 3

C H 3

S i

C H 3

C H 3

C H 3n

EXTRACTION OF HUMAN SKIN COMPOUNDS FOR LC

6

Open loops

Sonicate

15 min

10 ml Isopropanol

+ internal standard

RT

Close loopsWipe skin with 70% isopropanol

prior to sampling

Sample skin surface

1 hourStore loops in foil at 4°C

Thermal desorb each loop

directly in GC inlet liner

Back extract

MeOH:ACN

1:1

200 µl

Sonicate 15min at RT

Remove loop

Place vial with glass insert in LC autosampler

5 µl injection

Volatiles

GC

Non- volatiles

LC

Ankle

Wrists

RESULTS

HUMAN SKIN SURFACE CHEMICAL PROFILE

LCMS

7

GCMS

IMS: Wrist Day 1 Positive mode IMS: Ankles Day 1 Positive mode GCxGC: Wrist Day 1 GCxGC: Ankles Day 1

BIG DATA DILEMMA

8

GCXGC-TOFMS DATA PROCESSING

9

200 400 600 800 1000 1200 1400 1600

0

500

1000

1500

2000

2500

3000

Time (s)

3267 104 150 196

272 376 596

660

734

896

1039

1334

1480

1665

1925

2048

2432

2558

2721

30223131

33253490

3626

3811

39164098

4456 4640

4955

5083

5215

LECO Stat compare Software

Fisher Ratio

Similarity ≥ 80%

RI ± 35

Excel

VLOOKUP/ SUMIFS

1D RT ± 3sec

2D RT ± 0,3sec

Normalised, IS

Multivariate

Statistics

JMP

ANKLE VS WRIST (M=29), PCA SCORE PLOT

10JMP Pro 14

Reporting compounds with similarity >800

Tentatively identified

NIST14 library

-40

-20

0

20

40

Sample

20180626A Day1 MW Wrist a

20180626B Day1 MW Wrist b

20180626C Day1 MW Wrist c

20180626D Day1 MW Ankles a

20180626F Day1 MW Ankles c

20180627A Day2 MW Wrists a

20180627B Day2 MW Wrists b

20180627C Day2 MW Wrists c

20180627D Day2 MW Ankles a

20180627E Day2 MW Ankles b

20180627F Day2 MW Ankles c

20180628A Day3 MW Wrists a

20180628B Day3 MW Wrists b

20180628C Day3 MW Wrists c

20180628D Day3 MW Ankles a

20180628E Day3 MW Ankles b

20180702 Day3 MW Ankles c

20180702B Day4 MW Wrists a

20180702C Day4 MW Wrists b

20180702D Day4 MW Wrists c

20180702E Day4 MW Ankles a

20180702F Day4 MW Ankles b

20180702G Day4 MW Ankles c

20180703A Day5 MW Wrists a

20180703B Day5 MW Wrists b

20180703c Day5 MW Wrists c

20180703D Day5 MW Ankles a

20180703E Day5 MW Ankles b

20180703F Day5 MW Ankles c

20180704A Method blank a

20180704B Method blank b

20180704C Method blank c

-40 -20 0 20 40

Component 1 (6,76 %)

Ankle

Wrist

Method

Blank

11

UPLC-IMS-HDMS DATA PROCESSING

12

Data acquired: MassLynx

Data processed: UNIFI

Mass error: 20ppm

RT ± 0,2min

Drift time ± 0,5mseconds

Normalise, IS

Multivariate Statistics

EZinfo

Marker table

ANKLE VS WRIST (M=30), PCA SCORE PLOT

13-900

-800

-700

-600

-500

-400

-300

-200

-100

0

100

200

300

400

500

600

700

800

900

-1000 0 1000

t[2

]

t[1]

Single subject, multiple (n=5) sampling eventsESI+

Ankle (15 observations)Major Mix, QCMethod BlankPooled SamplesWrist (15 observations)

Major Mix2_pMajor Mix3_pMajor Mix4_pMajor Mix_p

Method Blanka_pMethod Blankb_p

Method Blankc_p

MW_A_Day1a_p

MW_A_Day1b_p

MW_A_Day1c_p

MW_W_Day1a_p

MW_W_Day1b_p

MW_W_Day1c_pPooled Samples2_p

Pooled Samples3_pPooled Samples_p

MW_A_Day2a_p

MW_A_Day2b_p

MW_A_Day2c_p

MW_W_Day2a_p

MW_W_Day2b_p

MW_W_Day2c_p

MW_A_Day3a_p

MW_A_Day3b_p

MW_A_Day3c_pMW_W_Day3a_p

MW_W_Day3b_p

MW_W_Day3c_pMW_A_Day4a_p

MW_A_Day4b_p

MW_A_Day4c_p

MW_W_Day4a_p

MW_W_Day4b_p

MW_W_Day4c_p

MW_A_Day5a_p

MW_A_Day5b_p

MW_A_Day5c_p

MW_W_Day5a_p

MW_W_Day5b_p

MW_W_Day5c_p

EZinfo 2 - Marker table ankle vs wrist named p2 (M3: PCA-X) - 2019-09-12 09:24:46 (UTC+2)

OPLS-DA

14

-1200

-1000

-800

-600

-400

-200

0

200

400

600

800

1000

-600 -500 -400 -300 -200 -100 0 100 200 300 400 500 600

t[2]O

t[1]P

Single subject, multiple day (n=5) sampling events

Ankle vs Wrist ESI+

Ankle

WristMW_A_Day1a_p

MW_A_Day1b_p

MW_A_Day1c_p

MW_W_Day1a_p

MW_W_Day1b_pMW_W_Day1c_p

MW_A_Day2a_p

MW_A_Day2b_p

MW_A_Day2c_p

MW_W_Day2a_p

MW_W_Day2b_p

MW_W_Day2c_p

MW_A_Day3a_p

MW_A_Day3b_pMW_A_Day3c_p

MW_W_Day3a_p

MW_W_Day3b_p

MW_W_Day3c_p

MW_A_Day4a_p

MW_A_Day4b_p

MW_A_Day4c_p

MW_W_Day4a_pMW_W_Day4b_pMW_W_Day4c_p

MW_A_Day5a_p

MW_A_Day5b_p

MW_A_Day5c_p

MW_W_Day5a_p

MW_W_Day5b_p

MW_W_Day5c_p

EZinfo 2 - MarkerTable_Ankle vs Wrist_p (M3: OPLS-DA) - 2019-09-11 09:37:09 (UTC+2)

S-PLOT

15

Wrist

Ankle

16

17

MARKERS

LC RESULTS

18

Progenesis QI v2.3

q-value ≤ 0.05

p-value ≤ 0.05

O

N

N,N-Diethylbenzeneacetamide

OH

O

OIsolinderanolide

H

H

H

H

H

HO

Abs

Campestanol

Ankle Wrist

O

O

O

P

O

OH

HO

DHAP

MARKERS

LC RESULTS

ID Class of

compound

Adduct Ret.

Time

(min)

m/z (Da) Collision

Cross

Section

(Å2)

Formula Mass Error

(ppm)

Isotope

Similarity

Fragments

(Peak m/z)Database Marker

CampestanolErgosterols and

derivativesM+Na 14.66 425.3764 216.715 C28H50O -3.19 90.86 - HMDB* Wrist

Isolinderanolide Tetrahydrofurans M+NH4 13.00 354.2992 202.09 C21H36O3 2.61 83.98 1 (312.2902) HMDB Ankle

Olomoucine 6-alkylaminopurines M+H 10.08 299.1615 175.489 C15H18N6O 0.098 88.01 - HMDB Ankle

N,N-

DiethylbenzeneacetamidePhenylacetamides M+H 6.59 192.1377 144.382 C12H17NO -2.87 95.86

2 (119.0488,

91.0570)HMDB Ankle

DHAP(18:0e)O-alkylglycerone

phosphatesM+H 7.38 423.2880 216.247 C21H43O6P 2.33 89.59 - HMDB Wrist

2-(Ethylamino)-4,5-

dihydroxybenzamide2-aminobenzamides M+NH4 6.59 214.1196 158.547 C9H12N2O3 4.90 96.07 - HMDB Ankle

2-Tetradecanone Ketones M+NH4 8.08 230.2469 178.356 C14H28O -4.21 92.60 - HMDB Wrist

19

* HMDB: Human Metabolome Database Version 4 created 10 July 2019

MARKERS SEPARATED BY DRIFT TIME

LC RESULTS

ID Class of

compound

Adduct Ret.

Time

(min)

m/z (Da) Collision

Cross

Section

(Å2)

Formula Mass Error

(ppm)

Isotope

Similarity

Fragments

(Peak m/z)Database

3β-Acetoxy-19α-hydroxy-

12-urseneTriterpenoids M+Na 11.21 507.3811

236.849

(Wrists)

210.679

(Ankle)

C32H52O3 0.41 79.34 - HMDB*

20

* HMDB: Human Metabolome Database Version 4 created 10 July 2019

O

HO

O

MULTIPLE SUBJECTS (N=20), SINGLE SAMPLING EVENT

GCxGC-TOFMS

Ankles and wrist

X2 biological

repeats

Ankles (m=39)

Wrists (m=40)

79 observations

21

UPLC-IMS-qTOFMS

Ankles and wrist

X3 biological repeats

Ankles (m=60)

Wrists (m=60)

120 observations

240 injections (ESI+ & ESI-)

22

Name

001Aa

001Ab

001Wa

001Wb

002Aa

002Ab

002Wa

002Wb

003Aa

003Ab

003Wa

003Wb

004Aa

004Ab

004Wa

004Wb

005Ab

005Wa

005Wb

006Aa

006Ab

006Wa

006Wb

007Aa

007Ab

007Wa

007Wb

008Aa

008Ab

008Wa

008Wb

009Aa

009Ab

009Wa

009Wb

010Aa

010Ab

010Wa

010Wb

011Aa

011Ab

011Wa

011Wb

012Aa

012Ab

012Wa

012Wb

013Aa

013Ab

013Wa

013Wb

014Aa

014Ab

014Wa

014Wb

015Aa

015Ab

015Wa

015Wb

016Aa

016Ab

016Wa

016Wb

017Aa

017Ab

017Wa

017Wb

018Aa

018Ab

018Wa

018Wb

019Aa

019Ab

019Wa

019Wb

020Aa

020Ab

020Wa

020Wb

Method Blank a

Method Blank b

Co

mp

on

en

t 2

(9,7

9 %

)

Ankles (m=39)

Wrists (m=40)

GCxGC

Ankles vs Wrists

20 Subjects, single sampling event

Component 1 (16,2 %)

23-10

-5

0

5

10Name

001Aa

001Ab

002Aa

002Ab

003Aa

003Ab

005Ab

008Aa

008Ab

009Aa

009Ab

010Aa

010Ab

011Aa

011Ab

012Aa

012Ab

013Aa

013Ab

014Aa

014Ab

015Aa

015Ab

017Aa

017Ab

018Aa

018Ab

019Aa

019Ab

Method Blank a

Method Blank b

-10 -5 0 5 10

Component 1 (14,5 %)

0

1

2

3

4

5

6

1 2 3 4 5

(5%); 1

(25%); 5 (25%); 5

(15%); 3

(30%); 6

1 = Not attractive

5 = Highly attractive

How attractive do you consider yourself to be

for mosquitoes?

(n = 20)

24

-10

-5

0

5

10

Oleic Acid

Ethanol, 2-(2-ethoxyethoxy)-

Nonane, 2,2,4,4,6,8,8-heptamethyl-

Octanal

2-Butoxyethyl acetate

n-Hexadecanoic acid

(2,3,5,6-Tetrafluorophenyl)methyl 3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropane-1-carboxylate

Octadecanoic acid, 2,3-dihydroxypropyl ester

Butyrolactone

Benzaldehyde, 4-methyl-

4-Cyanocyclohexene

2,6-Diisopropylnaphthalene

Ethanol, 2-(dodecyloxy)-

Octane, 1,1'-oxybis-

1,3,5,7-Cyclooctatetraene

Benzenemethanol, à,à-dimethyl-

Isopropyl palmitate

Hexadecane, 2,6,10,14-tetramethyl-

Naphthalene, 5-ethyl-1,2,3,4-tetrahydro-

Sorbic Acid

Pentadecane, 2-methyl-

Benzenebutanoic acid, ç-oxo-, ethyl ester

1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester

Pentadecanoic acid

Benzonitrile, 3,5-dimethyl-

Acetic acid, octyl ester

Hexadecane, 3-methyl-

Tetradecanoic acid

1-Heptanol, 6-methyl-

1-Octanol

Dodecanoic acid

Tridecanoic acid

Geraniol

9,12-Octadecadienoic acid (Z,Z)-

Phenanthrene

Name

001Aa

001Ab

002Aa

002Ab

003Aa

003Ab

005Ab

008Aa

008Ab

009Aa

009Ab

010Aa

010Ab

011Aa

011Ab

012Aa

012Ab

013Aa

013Ab

014Aa

014Ab

015Aa

015Ab

017Aa

017Ab

018Aa

018Ab

019Aa

019Ab

Method Blank a

Method Blank b

-10 -5 0 5 10

Component 1 (14,5 %)

25-8

-6

-4

-2

0

2

4

6

8Sample

001Wa

001Wb

003Wa

003Wb

004Wa

004Wb

005Wa

005Wb

006Wa

006Wb

007Wa

007Wb

008Wa

008Wb

009Wa

009Wb

011Wa

011Wb

013Wa

013Wb

014Wa

014Wb

015Wa

015Wb

017Wa

017Wb

018Wa

018Wb

020Wa

020Wb

Method blank a

Method blank b

-8 -6 -4 -2 0 2 4 6 8

Component 1 (27,5 %)

No 20

GCxGC

Wrists

-8

-6

-4

-2

0

2

4

6

8

Benzene, (1-ethyldecyl)-

Benzene, undecyl-

3-Butylisobenzofuran-1(3H)-one

Acetic acid, (3-methylbutoxy)-, 2-propenyl ester

1,2-Cyclohexanedicarboxylic acid, diethyl ester

Triethyl citrate

Naphthalene, 2,6-dimethyl-

Propoxur

Dodecyl acrylate

Phenanthrene

-8 -6 -4 -2 0 2 4 6 8

Component 1 (27,5 %)

3

Feet, ankles and legs

55%

Face and neck

5%

Hands, wrists and

arms

15%

I get bitten all over my

body

25%

Which part of your body gets bitten most by mosquitoes?

(n = 20)

26

0

2

4

6

8

10

12

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

CO

UN

T

NO

RM

ALI

SED

MEA

N P

EAK

AR

EA

COUMPOUNS DETECTED

Would you say that when outdoors you are the person who preferentially gets bitten by mosquitoes?

Yes

No

Count (yes, n=10)

Count (no, n=10)

27

-800

-700

-600

-500

-400

-300

-200

-100

0

100

200

300

400

500

600

-300 -200 -100 0 100 200 300

t[2

]O

t[1]P

UPLC-ESI+

Ankles vs Wrist

(m=120)

AnklesWrists

EZinfo 2 - MarkerTable_Skin RWS_p (M3: OPLS-DA) - 2019-09-11 10:43:55 (UTC+2)

-1.0

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

-0.2 -0.1 -0.0 0.1

p(c

orr

)[1

]P (

Co

rre

latio

n)

p[1]P (Loadings)

S-Plot (Wrists = -1, Ankles = 1)

299.11043_5.530_3.54

192.14076_6.333_2.66

304.30064_9.222_4.40

365.13552_9.941_3.76

399.25075_10.063_4.70

421.23185_10.089_4.70332.33108_10.312_4.74

301.14103_10.355_3.46

149.02306_10.358_1.88437.20961_10.369_3.76

383.32032_10.371_4.42 383.20660_10.374_4.52

284.33159_10.441_4.48

531.35063_10.725_5.70

227.19983_10.742_2.95

487.32398_10.743_5.39443.29743_10.750_5.11

326.37865_10.758_4.89

429.31812_11.330_4.63385.29201_11.385_4.37

341.26627_11.418_4.06

340.28385_11.485_3.93

487.36016_11.810_4.93

398.23517_11.863_4.96

311.16587_11.993_3.59

477.22486_12.058_4.44

278.21276_12.119_3.91161.06024_12.155_2.07

256.26287_12.165_3.51

EZinfo 2 - MarkerTable_Skin RWS_p (M4: OPLS-DA) - 2019-09-11 10:50:36 (UTC+2)

-800

-700

-600

-500

-400

-300

-200

-100

0

100

200

300

400

500

600

-400 -300 -200 -100 0 100 200 300 400

t[2

]O

t[1]P

UPLC-ESI+

1-24-5

001_Aa_p001_Ab_p

001_Ac_p

002_Aa_p002_Ab_p

002_Ac_p

003_Aa_p

003_Ab_p

003_Ac_p

005_Aa_p

005_Ab_p

005_Ac_p

008_Aa_p008_Ab_p

008_Ac_p

009_Aa_p

009_Ab_p009_Ac_p

010_Aa_p

010_Ab_p

010_Ac_p

011_Aa_p

011_Ab_p

011_Ac_p

012_Aa_p 012_Ab_p012_Ac_p

013_Aa_p013_Ab_p013_Ac_p

014_Aa_p014_Ab_p014_Ac_p

015_Aa_p015_Ab_p

015_Ac_p

017_Aa_p017_Ab_p

017_Ac_p

018_Aa_p018_Ab_p018_Ac_p

019_Aa_p019_Ab_p

019_Ac_p

EZinfo 2 - Marker table Skin RWS named p scale (M4: OPLS-DA) - 2019-09-12 11:49:37 (UTC+2)

28

0

2000

4000

6000

8000

10000

Attractive Not attractive

Variable Averages 192.22142_6.331_2.70192.14076_6.333_2.66214.12023_6.333_3.25229.08758_8.612_2.71399.25075_10.063_4.70301.14103_10.355_3.46227.19983_10.742_2.95326.37865_10.758_4.89385.29201_11.385_4.37614.48346_11.621_6.48531.38650_11.784_3.42487.36016_11.810_4.93443.33340_11.839_2.99399.30682_11.859_2.82149.02462_11.863_1.93398.23517_11.863_4.96278.21276_12.119_3.91161.06024_12.155_2.07

EZinfo 2 - Marker table Skin RWS named p scale (M4: OPLS-DA) - 2019-09-12 11:52:55 (UTC+2)

0

1

2

3

4

5

6

1 2 3 4 5

(5%); 1

(25%); 5 (25%); 5

(15%); 3

(30%); 6

1 = Not attractive

5 = Highly attractive

How attractive do you consider yourself to be

for mosquitoes?

(n = 20)

29-700

-600

-500

-400

-300

-200

-100

0

100

200

300

400

-700 -600 -500 -400 -300 -200 -100 0 100 200 300 400 500 600 700

t[2

]O

t[1]P

UPLC ESI+Ankles

Feet, ankles and legs

Hands, wrist and arms

EZinfo 2 - Marker table Skin RWS named p body parts ankles2 (M4: OPLS-DA) - 2019-09-12 12:07:08 (UTC+2)

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

Ave

rage

Resp

onse

Markers

Feet, ankles and legs

55%

Face and neck

5%

Hands, wrists and

arms

15%

I get bitten all over my

body

25%

Which part of your body gets bitten most by mosquitoes?

(n = 20)

30

CONCLUSION & FUTURE WORK

Data integrity

Good chromatography (mass spec or UV detector)

Mass accuracy

Additional features: 2D GC or ion mobility

Collaboration with data scientist/ statisticians

Develop tools

Tentatively identify lead compound for vector control strategies

Differences between skin areas sampled

Differences between attractive vs not-attractive groups31

ACKNOWLEDGEMENTS

I would like to thank the following people for their support with the project:

Dr Hubert Mandery for financial support in his private capacity

Prof Leo Braack, UP ISMC

David Masemula (UP) for assembling the PDMS loops

Microsep, Tyren & Caryn

L’Oréal-UNESCO For Women in Science Sub-Saharan African Programme

32

Ethics approval: EC171109-159

33

"For Africa to move forward, you've really got to get rid of malaria."

— Bill Gates

THANK YOU!

QUESTIONS?