RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
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: [email protected]
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.
