RESEARCH POSTER PRESENTATION DESIGN © 2015

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Human milk is regarded as the gold standard for infant feeding.

INTRODUCTION

EXPERIMENTAL

CONCLUSIONS

[1] Why Human Milk Oligosaccharides Are the Next Big Breakthrough? www.nutrition news.abbott

Connie A. Remoroza1, Yuxue Liang1, Tytus D. Mak1, Sergey L. Sheetlin1, Yuri A. Mirokhin1, Joice San Andres2, Michael L. Power3, Stephen E. Stein1

1Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg MD 208992Central Luzon State University, Maharlika Highway Nueva Ecija 3120, Philippines

3Nutrition Laboratory, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, D.C., USA

TANDEM MASS SPECTRAL ANALYSIS OF FREE OLIGOSACCHARIDES IN MILK OF VARIOUS MAMMALS

Human milk oligosaccharides feed good bacteria in the gut

where 70% of the immune system exists [1].

This study focuses on the identification and molecular fingerprinting ofoligosaccharides derived from the milk of mammals using the Hydrophilic InteractionLiquid Chromatography and Mass Spectrometry for building NIST prebiotics/glycansMS library.

Human milk

SRM 1953

➢Removal of lipids & proteins

Sample reduction & clean up

➢SPE removes salts. 20% and 40% acetonitrile separates neutral and acidic oligosaccharides.

Analysis of HMOs & BMOs by HILIC-

ESI-MS

➢Identification of neutral and acidic oligosaccharides.

Molecular structure analysis using spectral library and SIM Glycan

Building of NIST oligos MS Library

NIST SRMs

Nomenclature of oligosaccharide

RESULTSI. Separation based on size, net charge & type of oligosaccharides

II. Data analysis using different analytical platforms

NIST Tandem MS Library

Glucose

Galactose

GlcNAc

Fucose

NeuAc

➢High resolution MS2

values

➢negative and positive mode

➢Different collision energies

➢Different adducts

➢Score or match factor (MF) provides the degree of structure similarity.

➢Error tolerance of below 10 ppm.

➢ In summary, 140 of humanmilk oligosaccharides andover 70 non humanoligosaccharides have beenidentified in milk of variousmammals.

➢ The HILIC-ESI-MS method incombination with NIST MSsearch engine can now beused to accurately determinevarious fucosylated andsialylated oligosaccharidespresent in infant formula andother domestic animal milk.

Email address: concepcion.remoroza@nist.gov

III. Hybrid MS Library Search facilitates the identification of unknown oligosaccharides

HILIC MS profile of oligosaccharides present in African lion milk.

IV. Quantitative analysis of oligosaccharides in milk of various mammals

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34Time (min)

Iso

glo

bo

trio

se

LNn

H

N30

20

LNn

T

LDFT

N40

10

LNFP

III

N60

20

N50

20

N51

20a

N51

20b

N41

30

N50

20

511.350.00 0.000.00

2’-F

L

3’-F

L

N11

10

N40

00b

N40

00a

N40

00 c

LNFP

III

Re

lati

ve

Ab

un

da

nc

e

3’-S

L 3’-G

L

6’-G

SL

3’-G

DL

3’-D

SL

DP 3 DP 4 DP 5 DP 6 DP 7 DP 8

N41

20

672.510.00 0.0 0.000.00 0.000.000.00 0.000.00 0.00 0.00 0.00 0.000.00

0.00

6’-S

L

3’-G

SL

N20

10

6’-D

SL

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

N31

20

N42

20

N40

20

3’-

GL

African Lion milkGoat milk

Buffalo milk

Bovine milk

Goat milk

Buffalo milkBovine milk

A B

CD

Isotopic masses

Isotopic masses

Monoisotopic mass

Monoisotopic mass0

20

40

60

80

100

3'-SL 6'-SL 3'-GL 6'-GL 3'-SLN A3001 A4001 a A4001 b A3011 a A3011 b A3002 S-LNnH

Rel

ativ

e ab

un

dan

ce

Lion Goat Buffalo SRM1549a

α2-3β1-4

α2-6β1-4

α2-6β1-4

β1-4

α2-3

β1-4

α2-3β1-4

β1-4

α2-3

β1-3 α1-4

α2-

β1-3 α1-4

β1-4

β1-3

β1-4

β1-6

β1-4

α2-6

β1-4

B

β1-4

0

20

40

60

80

100

Rel

ativ

e ab

un

dan

ce

Lion Goat Bufallo SRM1549a

A

β1-4

α1-2

β1-4

α1-3

β1-4

α1-3 β1-4

β1-3 β1-4

β1-6 β1-4

b1-4a1-3

β1-3 b1-4

β1-4

β1-4

β1-6

Smithsonian National Zoo Milk Repository

DeltaMass= 41.027

sMF score = 50

hMF score = 922

m/z 730.2378 SRM 1953 Glycan 3,4,3-GalactotetraoseHead to Tail MF=922 RMF=931

210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720

0

50

100

50

100

255.2315

275.0736

305.0840 347.0947

365.1055

365.1049

388.1214

406.1317

406.1316

429.3186

437.1251

500.2826

527.1575

550.1741

568.1842

599.1783

610.1959

640.2050

670.2163

671.1996

712.2268

712.2263

509.1470

0,2A4

Y2

B3

Y3

2,4A42,4A2

C2

B2

Z2

B1

2,4A4B2

Y2

B3

Z2

2,4A2

0,4A4

Y3

Z2

Y2B3 Y3

B3

Y2

B2

C

D

m/z= 878.3116 SRM 1953 Glycan LNFP IIIHead to Tail MF=826 RMF=855

300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870

0

50

100

50

100

289.0898367.1214

388.1204

450.1585

534.1795

550.1729

638.2261

670.2146

696.2323

696.2303

732.2533

756.2506 816.2723

818.2886

858.2824

860.2980

DeltaMass= 2.016

sMF score = 309

hMF score = 826

Y2

Y3a

B3

A

B

Y3

Y2

B2 /Y3

730.2357

Y2

Y3a

B3B2

B2

Y3

B3

0,4X0

2,4A4B3 /Y3

552.1901

- 18 Da

- 18 Da

Reduced

B2

Human milk reference material ➢Ion plot of identified compounds. This demonstrates the robust identification of unknown compounds using tandem mass spectral libraries.

➢Comparison of spectra based on MS2 similarity between oligosaccharides by IsoPique.