High-Throughput Milk Oligosaccharide Analysis Using a Rapid Cartridge-Based

Capillary Electrophoresis Instrument

Elisha GoonatillekeChemistry Department

University of California, Davis

Glucose

Galactose

N-Acetylglucosamine(GlcNAc)

N-AcetylneuraminicAcid (Neu5Ac)

Fucose

Human Milk Oligosaccharides (HMO)

Fucosylated54%

Sialylated7%Fuc +Sialyl

5%

UndecoratedNeutrals

34%

Ruhaak et al, 2012

HMO Class Percentages in breast milk

What is so beneficial about HMOs, if they are indigestible in the infant gut?

• Sialylated HMO may promote post-natal brain development.

Nutrition Reviews, Vol. 67 (Suppl. 2), S183-S191

HMO Functions

Infant protection by inhibiting binding of pathogens to epithelial surface

Prebiotics for beneficialbacteria in the infant’s gut.

Newburg et al 2007

HMOHealthy Bacteria

13%

7%

8%

10%

4%5% 6%

3%2%

2%2%2%2%

Courtesy of Dr. Jasmine Davis

LNT

2’FL

LNFP I

LNnT

LDFTMFLNH III & MFLNH I

LNFP II

DFLNHa

LSTc

IFLNH I

3’SL

LSTb

Human Milk Oligosaccharides Composition

• ABH antigens secreted in bodily fluids

• Indicator for being a secretor is α1-2 linked Fucose

• Typically around 20% of population are non-secretors

D. S. Newburg, G. Ruiz-Palacios, A. L. Morrow, Human Milk Glycans Protect Infants against Enteric Pathogens. Annu. Rev. Nutr. 25, 37 (2005).Brooks, S. A.; Dwek, M. V.; Schumacher, U. Functional and Molecular Glycobiology; BIOS Scientific Publishers Ltd: Oxford OX4 1RE, UK, 2002.

Nutrition Reviews, Vol. 67 (Suppl. 2), S183-S191.

OHOHO

ONHAc

ORO

OHOH

HOO

O

HOOHOH

H DeterminantSecretorα1-2

Importance of Secretor Status

Jasmine Davis, Sarah Totten

Secretor Non-secretor

Lower instances of diarrhea X

Promotion of a healthy microbiota X

More protection against bacterial infections & autoimmune diseases X

Higher amounts of total oligosaccharides X

More resistant to norovirus, influenza, rhinovirus, and HIV-1 X

Lower rate of several common digestive tract cancers X

Separation

High pH Anion-Exchange Chromatography

Capillary Electrophoresis

Liquid Chromatography

Detection

Fluorescence Detection

Ultraviolet detection

Mass Spectrometry

Nuclear Magnetic Resonance

Methods for detection and quantification of Oligosaccharides

Gly-Q

Capillary Electrophoresis

1800

1809: Ferdinand Frederic Reuss suspended clay particles in an aqueous solution to migrate under the influence of an electric field

1950: Gel electrophoresis1981: James Jorgenson demonstrated that the use of capillaries overcome many of the

heating issues and stabilizing media are no longer necessary1989: Developed the first commercial instrument

Currently capillary electrophoresis is used for analysis of Nucleic acids, Proteins, Metabolites, Pharmaceuticals and Carbohydrates

1900 2000

Why develop methods to analyze oligosaccharides using Gly-Q ?

Anionic Label

OS Sample

Gly-Q: Capillary electrophoresis in a cartridge

Signal

Separation occurs less than 2 minutes

Gly-Q Manager Software • DP2 and DP15 standards co-injected with samples for alignment• GU ladder injected in sequence to automatically generate GU

values

Gly-Q: Data AnalysisElectropherogram

DP2

DP15DP2DP15

Alignment of peaks

• Peak assignment using GU values

Gly-Q instrument

Replaceable cartridge with gel matrix & capillary

Features

Throughput 2 x 96 well plates per day

Analysis time per sample <2 minutes per sample

Injection Electrokinetic(Virtually no sample consumption like CE)

Detection fluorescence detection

Ease of use Click-in capillary, 96-well plate-compatible

Instrument Footprint 15”x16”x12” (L x H x W) 38cm x 40cm x 30cm

Gly-Q: Characteristics

Profiling Milk Oligosaccharides

Milk oligosaccharides

Labeled oligosaccharides + dye

•APTS label

Labeled Oligos

Data

2 minute Gly-Q experiment

Milk

Protein & lipid removal

Label

Cleanup

Sample analysis

• Centrifugation• Ethanol precipitation• Solvent dry down

•HILIC

Human Milk Oligosaccharide Analysis Workflow

0.6 1.00.8 1.2Time (min.)

Sign

al (%

)

Gly-Q Analysis of HMOs

Sample 1

Sample 2

Sample 3

Sample 4

LNT

LNnT

LNnH

LNFP I

LNFP III + LNFP V

MFLNH III

3’SL6’SL

3’SLN

6’SLN

LSTa

LSTb

LSTc

DSLNT LNDFH I

LNDFH II

2’FL

3FLLDFT

DP2

HMO Standards Analyzed with Gly-Q

Oligosaccharide GU Value

6’SL 2.57

6’SLN 2.68

3’SL 2.95

3’SLN 3.11

DSLNT 3.73

3FL 3.96

2’FL 4.01

LSTc 4.03

LSTa 4.21

LSTb 4.42

LDFT 4.61

LNnT 4.84

LNT 5.11

LNFP I 5.90

LNDFH I 6.01

LNFP V 6.07

LNFP III 6.08

LNDFH II 6.36

LNnH 6.49

MFLNH III 7.25

GU Library with Oligosaccharide Standards

• GU library is used to automatically label the oligosaccharide peaks

Secretor Status

3’SL6’SL

6’SLN

Lactose

DSLNT

3FL and 2’FL

LNFP III + LNFP V

LNFP I LNDFH I

LNT

LNnT

LDFT

LSTb

Sign

al (%

)

0.6 0.80.7 0.9 1.0Time (min.)

Secretor 1

Secretor 2

Secretor 3

LNFP II

Profiles of Secretor Mother’s Milk Oligosaccharides

3’SL6’SL

6’SLN

Lactose

DSLNT

3FL and 2’FL

LNFP III + LNFP V

LNFP I LNDFH I

LNT

LNnT

LDFT

LSTb

Sign

al (%

)

0.6 0.80.7 0.9 1.0Time (min.)

Profiles of Non-Secretor Mother’s Milk Oligosaccharides

Non-Secretor 1

Non-Secretor 2

Non-Secretor 3

3’SL6’SL

6’SLN

Lactose

DSLNT

3FL and 2’FL

LNFP III + LNFP V

LNFP I LNDFH I

LNT

LNnT

LDFT

LSTb

Secretor

Non secretor

Sign

al (%

)

0.6 0.80.7 0.9 1.0Time (min.)

Identification of Secretor Status

Nano-HPLC- Chip/TOF MSdata of Secretor Status

Quantification of Oligosaccharide in Bovine Milk

Whey permeate

Still contains OS, Lactose, minerals

Bovine milk OS concentrate

Sample A Sample B

Industrial process

1-1.5 g/L OS in colostrum, 0.1-0.2 g/L OS in mature milkLess complex than HMO

Diverse dairy products

Bovine Milk

Bovine Milk Oligosaccharide Analysis Workflow

Labeled oligosaccharides + dye

•APTS label

Labeled Oligos

Data

2 minute Gly-Q experiment

Milk Permeate

Label

Cleanup

Sample analysis

•HILIC

Internal StdDP7

3’SL

6’SL

6’SLN

DSLNTLSTc

2’FL

3FL

Lactose

6SL0% 3SL

2%

Lac98%

LNT0%

Electropherogram of Sample A (Whey Permeate)

6SL9%

6SLN5%

3SL69%

3SLN12%

Lac0%

2FL, 3FL, LSTc2%

LSTa2%

LNnT1%

3’SL

6’SL

6’SLN

3’SLN

DSLNT

LSTc

2’FL

3FL

LSTa LDFT

LNnT

Electropherogram of Sample B (OS Concentrate)

Whey permeate Bovine milk OS concentrate

Sample A Sample B

Industrial process

6SL9%

6SLN5%

3SL69%

3SLN12%

Lac0%

2FL, 3FL, LSTc2%

LSTa2%

LNnT1%

Electropherogram of Sample A (Whey Permeate)

6SL0% 3SL

2%

Lac98%

LNT0%

0

5

10

15

20

25

30

6SL 6SLN 3SL Lac LNnT

[Olig

osac

char

ides

](g

/100

g)

Gly-Q

Dionex

Whey permeate Bovine milk OS concentrate

Sample A Sample B

Industrial process

Comparability of Gly-Q to Dionex Method

0

10

20

30

40

Lac

[Lac

tose

] (g/

L)

Gly-Q

Dionex

0

0.2

0.4

0.6

0.8

6SL 3SL LNT[Olig

osac

char

ides

] (g

/L)

Gly-Q

Dionex

N= 6

N= 6

N= 6

Conclusion

A rapid and sensitive methods were developed for quantitativeand qualitative analyses of oligosaccharides using Gly-Q.

Sample preparation is simple, rapid and automatable

Fast separation ( < 2 min)

Automated data analysis

Suited for large number of sample analyses

Acknowledgements

Lebrilla GroupDr. Carlito LebrillaDr. John K. MuchenaDr. Mariana Barboza

Gege XuMatthew Amicucci

Ace GalermoQiongyu Li

Maurice WongEshani Nandita

Barile GroupDr. Daniela Barile

Dr. Jaime Salcedo

Dr. Andres GuerreroDr. Jasmine Davis

CollaboratorsDr. Jennifer Smilowitz