The document summarizes research analyzing the types and percentages of free oligosaccharides in human breast milk from mothers in Bangladesh. Using mass spectrometry, the researchers identified both neutral and sialylated oligosaccharides. They are creating an in-house database of human milk oligosaccharides (HMOs) to more efficiently analyze remaining samples. The database will contain HMO structures organized by core and account for modifications. It is hoped that the database will help reduce manual data analysis and allow investigation of structures over 5000 Daltons.

1. Structure of Free Oligosaccharides in Human
Breast Milk
Arjuna Karikaran, Sara Porfírio, Stephanie Archer-Hartmann, Parastoo Azadi
Complex Carbohydrate Research Center (CCRC), UGA, Athens, GA
Abstract
• The purpose of the research was to determine the type and relative
percentages of free oligosaccharides that were found in human breast milk
from mothers in Bangladesh.
• Bangladesh was chosen because the study wishes to see how malnutrition
of mothers translates into changes in the structure of the oligosaccharides
produced by the mother. Specifically, the ratio of sialylated milk
oligosaccharides as they are known to promote better growth of infant
microbiota.
• Using the Fusion Orbitrap MS instrument, we have performed LC-MS/MS
analysis on permethylated human milk samples and so far have found both
neutral and sialylated oligosaccharides, including isomeric forms.
• Without a functioning database, analysis of LC-MS/MS data of milk samples
is done manually - a very tedious and involved process.
• We are currently creating an in-house database of human milk
oligosaccharides (HMOs) that we can use to analyze the remaining samples
more efficiently.
Methods: GlycoWorkBench (GWB)
1. Glycoworkbench is a software tool that allows for modeling of the HMOs
that are being studied
2. Lists of known HMOs structures were sourced from the literature (Chen,
2015; Orsel 2017)
3. The HMOs were grouped according to “cores”
4. Cores are based on the Galactose and N-acetyl glucosamine (GlcNAc)
backbone that is common to many structures (Figures 3-8)
5. Variations on each core result from fucosylation and sialylation
6. Modifications such as derivatization (ex: permethylation) and mass adducts
(ex: sodiated forms of HMOs) can be accounted for
Results: MS/MS fragmentation of a fucosylated HMO
Fig. 1 MS2 spectrum of a small molecular weight, neutral HMO (m/z 1549.77).
Several isomers (position and linkage) can be identified for this glycoform. MS/MS
fragmentation patterns aid in structure assignment. A possible Core for some of
these isomers is shown to the right (Fig. 3).
References
Chen, X. (2015). Human Milk Oligosaccharides (HMOS). Advances in Carbohydrate Chemistry and Biochemistry, 72:113-190. doi:10.1016/bs.accb.2015.08.002
Mcguire, M. K., et al (2017). What’s normal? Oligosaccharide concentrations and profiles in milk produced by healthy women vary geographically. The American Journal of Clinical Nutrition 105(5):1086-1100. doi:10.3945/ajcn.116.139980
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Conclusions and future work
Core Examples
Core VIIICore V Core VII
Fig. 3 Examples of different HMOs core structures (top) and their decorated
counterparts (bottom). Fucoses are represented as red triangles and can be
attached in α-2, 3, or 4 linkages. Sialic acids are represented by purple
diamonds and can be attached in α-3 or 6 linkage.
Non-fucosylated, non-sialylated core structures
Fucosylated and sialylated derivations of core structures
Methods: Database Creation
• Presently all GWB files created for each core of structures are undergoing a
final checking process to verify structure and linkage accuracy.
• Accuracy is important as it is hoped that this database will become the in-
house reference for all HMOs work done in the future.
• Once finished, this database will help reduce considerably the amount of
manual data analysis of HMOs LC-MS/MS datasets.
• Future work includes updating the database if new HMOs structures are
discovered, as well as clarifying structures that are ambiguous at the
moment.
• Other future work includes investigating the effects of using different
matrices in MALDI-TOF-MS analysis of HMOs, aiming at increasing the
molecular weight range of this analysis.
• We hope this study will allow the analysis of structures with molecular
weight above 5000 Daltons.
Gather literature reported mass list
Create structures on GWB (including
linkages)
Organize structures by core
Save GWB files with appropriate
settings (ex: permethylation, charge
state, adduct type)
Final Database creation
2708.15796 -0.192 (Hex)5(HexNAc)3(Deoxyhexose)3(NeuAc)1
2721.16943 -0.176 (Hex)5(HexNAc)3(Deoxyhexose)1(NeuAc)2
2796.19629 -0.206 (Hex)6(HexNAc)4(Deoxyhexose)3
2895.24072 -0.194 (Hex)5(HexNAc)3(Deoxyhexose)2(NeuAc)2
2898.23828 0.788 (Hex)7(HexNAc)5(Deoxyhexose)1
2970.26611 -0.226 (Hex)6(HexNAc)4(Deoxyhexose)4
3069.28223 -0.241 (Hex)5(HexNAc)3(Deoxyhexose)3(NeuAc)2
3071.30029 -0.239 (Hex)7(HexNAc)5(Deoxyhexose)2
3245.3811 -0.248 (Hex)7(HexNAc)5(Deoxyhexose)3
Results: MS/MS fragmentation of a fucosylated and sialylated HMO
Include
fragments
Top row
with labels
500 600 700 800 900 1000 1100 1200 1300 1400 1500
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
1086.5312
z=1
1313.6475
z=?
1550.7765
z=1
912.4423
z=1
1343.6579
z=1
660.3211
z=? 1239.6108
z=1
1139.5576
z=1
880.4160
z=?
806.3790
z=?
486.2314
z=? 676.3156
z=1
676.3160
z=? 1361.6678
z=?
1109.5471
z=1
or
or or or
oror
or
oror
or or
or oror
oror or
Structures
[M+Na]+
Several different isomers can be identified for this mass
*
*
*
*
*
* *
or or or
400 600 800 1000 1200 1400 1600 1800 2000
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
1496.7353
z=2
1005.4869
z=3
1453.7164
z=2
976.8073
z=3
1052.5126
z=3
1567.2743
z=2
1366.1668
z=2
1309.1398
z=21091.5331
z=2660.3216
z=1486.2316
z=1 1168.0735
z=2
1685.3363
z=2398.1791
z=1
834.4081
z=1620.2904
z=? 1956.9689
z=?
[M+Na]2+
30 32 34 36 38 40 42 44 46 48 50
Time (min)
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
39.05
41.38
45.20
36.38
As glycoform complexity increases, the number of
possible isomers increases proportionately. Although
some resolution is observed (several peaks are
visible in XIC - inset), separation conditions need to
be improved in order to fully resolve these
structures.
HMO isomers
XIC of m/z
1030.2140
(z=3)
Distinguishing fragments
or
Fig. 2 MS2 spectrum of a large molecular weight, fucosylated, sialylated HMO (m/z
3344.66). Structure assignment becomes more difficult with increased molecular
weight. More MS/MS (MSn experiments) data is needed to determine accurate
structures. A functioning database could help exclude non-biologically relevant
structures.
Core VIIICore V Core VII
Monosacch
aride code