Carlito B. LebrillaDistinguished Professor

Department of Chemistry

Department of Biochemistry and Molecular Medicine (School of Medicine)

University of California, Davis

Many functions of human milk

Lactose

(Glyco)Lipids

Oligosaccharides (HMO)(Glyco)Proteins(Glyco)Peptides

Cells

RNADNA

MetabolitesMinerals Microbes

Micelles

Components of milk

Nutrition Microbiota

Development

Infant Development

Protection

Early studies on milk has focused on nutrition.Milk contains functional compounds that can have long term consequences to health.

Milk is highly glycosylated

Developed and employed tools for Oligosaccharide analysis

Oligosaccharides are an analytical challenges:Developed many tools to characterize and quantitate

glycosylation in tissue

Lactose

(Glyco)Lipids

Oligosaccharides (HMO)(Glyco)Proteins(Glyco)Peptides

Cells

RNADNA

MetabolitesMinerals Microbes

Micelles

Components of milk

Milk is highly glycosylatedProteomicGenomic Glycomic

HMO library : at least two phenotypes to human milk

Davis et al – ManuscriptWu et al. JPR 2012Wu et al. JPR 2012

Fut2 Gene in epithelial cells

Lewis b+ Milk type Secretor

Lewis b- Milk type Non Secretor

Analysis >5000 milk samples

Overlay >100 chromatogramMethod for determination

highly reproducible.

Wu et al. J. Proteome Res. 2010, Wu et al. J. Proteome Res. 2010.

Antigens in HMO similar to blood type antigensBased on the analysis of milk, structures yielded

biosynthetic pathways.

HMOs include many antigens and receptors

Human milk oligosaccharides are similar to blood group determinants

Binding site to pathogens

LC-MS profile of HMOA library of HMO structures produced

to understands role of human milk oligosaccharide

Totten et al. J. Proteome Res. 2012

10uL of human milk Two methods for AnalysisnanoLC chip –QTOF MS

UPLC–QQQ MS

Like Counting Hippopotamuses in river of crocodiles

Hippo out of the water(NanoLC - QTOF MS)Convince hippo to leave the waterNot all the hippos may leave No mistaking hippo for crocsCan tell hippos apart

Count the Hippo in the water(UPLC-QQQ MS)May mistaken crocodiles for HippoHard to to tell the size of the hippoHard to tell one hippo from another

Two methods for profiling HMO

NanoLC - TOF MSUPLC – QQQ MS

UPLC – QQQ MS• Absolute quantitation (mg/mL)• No enrichment• Less specificity

• High throughput• >200 samples/day

NanoLC – MS TOF• Quantitation in ion counts• Enrichment necessary to quantitate• Better Specificity• More compounds >300• Low Speed • >24 samples/day

Methods for profiling HMO

HPLC – UV• 1 hour analysis• Widely available, Previous

standard• Absolute quantitation• <30 compounds

Requires chemical labelingDifferent reactivitiesCompleteness of reactions (not

100%) – limits dynamic range Requires commercial standards

ExpensiveVery impure Variations in retention times

Compounds misidentifiedDifficult to separate class

MALDI MS (No isomer separation)

CE– Fluorescence• <1 minute analysis• Absolute quantitation• <30 compounds

Requires chemical labelingDifferent reactivitiesCompleteness of reactions (not 100%) Requires commercial standards

ExpensiveVery impure Variations in retention times

Compounds misidentified

HPLC-QTOF MS• 1 hour analysis• Relative quantitation• >300 compounds• Discovery

Requires chemical reduction Differential ionization will

yield different responses

UPLC –QQQ MS• 10 minute analysis• Absolute quantitation• < 30 distinct compounds

(separate by class)

Requires standards

Separation

MALDI Probe

Factors that affect the abundances of HMO• Sex of infant• Age of Mother• Number of children• Gestational Diabetes• Days postpartum• Term Delivery• Secretor Status• Diet

Xu et al J Nutrition 2016

Absolute quantitation using MRM (LC-QQQ) shows: Decrease in absolute abundances

during lactation. Decrease in abundances of

fucosylated structures. Decrease in abundances of

sialylated structures Secretors have more HMO,

Fucosylation, less sialylation

UC Davis CohortJennifer Smilowitz, J. Bruce German

UPLC –QQQ MS

0.00

0.25

0.50

0.75

1.00

1.25

0 1 2 3 4 6 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 1 2 3 4 5 6 0 1 2 3 4 5 6

United States Brazil Peru South Africa India Bangladesh

• Error bars given as standard deviation• Order of country (left to right) decreasing GDP (per capita, based on 2014 United Nations rankings)• Note that United States, Peru, South Africa, and Bangladesh have a month 0 time point• Note that United States has no month 5

CountryLactation Month

MAL-ED:Total HMO Decreases Throughout Lactation

Diet affects HMO production

• A swing of around 5 kg/year, mostly lost from stored body fat.

• Food sources begin to deplete in wet season, food becomes less available.

• Heavier work load, expending more energy than taking in.

The affect of large diet change on HMO was observed in studies in The Gambia.

Diet affects HMO production

Nearly all Infants rich in Bifidobacterium

Production of HMO during birth and lactating season.

Angela Zivkovic

Davis et al Under Revision 2016

Most infants rich in Bifidobacterium.Mothers lactating through wet season produce less HMO.

David Mills

HMO decreased during wet “hunger” season

Diet affects HMO production

Structures correlated with child morbidity Infants with sick days had less LNFP1, more LNT.

Angela Zivkovic

Mother self reported.“Sick” – Any day with illness (cold, diarrhea, etc.)“Not Sick” – No illness days reported.

Sample Preparation/Extraction

Project Sub-Project Description Sample Type # of Samples HMOs Glycopeptides Peptides Protein Measurement N-glycans

Gat

es/B

MM

I

LCNI-5/Malawi

Healthy vs Stunted, extreme cases

Milk 117+12 X X X X

Feces 54 X X (free)

Lactation/ Davis

Timepoints (2-347 days)Control Study

Paired Milktest set 107 X

Paired Feces 107 X X X (free)

Milk 516 X X X (n=92)MAL-ED

-India-Peru

-South Africa-Brazil

-Bangladesh

Timepoints(0-6 Months)

Phenotypic bins of LAZ scores

(HH, HR, SG, SS)

Phenotypically Binned 280 X X

Non-binned 570 X X

iLiNS Dyad/Malawi

3 nutrient suppl3 Lactation Timepoints (1,3,6)Healthy Vs Severely Stunted Milk

259 (263 actual) X X

Stunted/Non-stunted/ extreme cases 536 (553 actual) X X

ReceivedProcessing

(extraction + instrument run)

Data AnalysisCompleted

Gates Fund Project on Environmental Entheropathy

Jeff GordonJ. Bruce GermanDavid MillsDaniela Barile

-Mothers of healthy infants had higher levels of total, fucosylated, and sialylated HMOs-Non-secretors mothers of stunted infants had significantly lower levels of HMO

< 3> 0

< 2> 0

Large Multisite Study on HMO and Stunting

Charbonneau et al Cell 2016

0.0005

0.001

0.0015

0.002

0.0025

0.003

3FL

Healthy Severely Stunted

LAZ bin

Specific structures correlate with stuntingAbsolute abundances

0.005

0.01

0.015

0.02

0.025

3'SL

Healthy Severely Stunted

LAZ bin

3FL 3’SL

P = 0.0192P = 0.0090

p-LNH

P = 0.0114

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

p-LNH

Healthy Severely Stunted

LAZ bin

LNFP II

P = 0.0067

3120

P = 0.0347

5130a

P = 0.0382

LNFP II

P = 0.0289

5130a

P = 0.0321

LDFT

P = 0.0499

3’SL

P = 0.4433’SL

3120

P = 0.0037P = 0.384

3’SL

3FL

p-LNH

LSTc

P = 0.0375LSTa

P = 0.0021

P = 0.0295

3FL

P = 0.0037

3FL

MFLNH III+MFLNH I

P = 0.0293*

TFLNH

P = 0.0154

Month 1

Month 3

Month 6

LNFP II

P = 0.0493

5130a

P = 0.034

Secretor

Non-Secretor

Glucose (Glc)

Galactose (Gal)

Fucose (Fuc)

N-Acetylglucosamine (GlcNAc)

N-Acetylneuraminic Acid (Neu5Ac)

Secretor Status - a factor in interaction with gut microbiota

Charbonneau et al Nature 2016

643

2

B+ Milk Type

B- Milk Type Secretor is more than just α(1,2)fucose

Distribution of Lewis B+/B- milk

Davis et al. - Manuscript

Katie Hinde (U. Arizona)Andrew Prentice (U. Cambridge)J. Bruce German (UC Davis)MAL-ED (BMGF) –Jeff GordonILYNS (BMGF) –Jeff Gordon

Combined > 1000 samples

Map of Secretor distribution based on mother’s milk.

Distribution of Lewis B+/B- milk

Davis et al. - Manuscript

Isolated tribes in South America

Africa – high levels of non secretors

Out of Africa model shows non-secretors diminishing.Nature 2016

US secretor more representative of Europe

South America Indigenous have little or no non-secretors.

Cholera Outbreaks Correlate with High Number of Non-secretors

http://gamapserver.who.int/mapLibrary/app/searchResults.aspx

Local diseases select secretor types

Cholera could affect infants more strongly than adults.

Cholera response affected by blood types.

Fleckenstein Am J. Trop Med 2016

HMO Found in other bodily fluids

MilkFeces

Urine

BloodMother

InfantHMO, modified HMO

HMO

HMO, HMO fragments, N-glycansBlood

J Bruce GermanMark Underwood

Infant Blood

Mother Blood

HMO Found in Infant Blood

Ruhaak et al. Anal Bioanal Chem 2014

Blood of infants yield HMO and BMO.

Exclusively formulaBreast + formula

• HMO found in blood of breast fed infants.

• BMO found in blood of formula fed infants.

• Distinguish between breast fed and formula fed infants.

HMO Found in Mother’s blood

Xu et al. Manuscript

• HMOs found in blood of lactating women ~1 ug/mL HMO in blood.

• BMO primarily found in blood of Men and nonlactating women.

• Some HMO found in blood of men and non-lactating women.

x 3

HMOs are prebiotic

Consumption patterns ofB. infantis strain

Sialidases - ------------------Sela et al JBC 2011Fucosidases - ---------------Sela et al AEM 2012Hexosaminidases - --------Garrido et al Anaerobe 2012Galactosidases - ----------- Garrido et al Food Micro 2012Surface Binding Proteins Garrido et al PLoS One 2011

Strum et al An Chem 2012

Enzymes found in B infantis cleave

HMO Bifido longhum sp infantis

David Mills

HMO in feces increase until commensals are established

DeLeoz et al. J Proteome Res 2014

Bifido longhum sp infantis

HMO builds up in feces until a sufficient population of saccharolytic bacteria populates the gut.

The abundance of HMO in feces decreases significantly when this condition is met.

Feces contain glycans from glycoproteins

Feces contain glycans from glycoproteins

Davis et al. Molec Cellular Proteomic 2016

Digested N-glycansDigested HMO

Tandem MS (MS/MS) used for structural elucidation.Structures correspond to digested HMO and digested N-glycans.HMO and Glycoproteins sources of prebiotic!

Feces contain glycans digested by B. Infantis enzymes

Breast milk

Feces

B-galactosidaseBLNG_00015

Identify structures

Digestion of HMO with expressed enzymes yielded the structures in feces.Collective enzymes in the gut biota produces feces products.

BLNG_00015 PNGAse F EndoBI-1

Quantification of monosaccharides using 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization

We can follow the degradation to the monosaccharides.New quantitation method mates well with HMO analysis.

LC-MS method for monosaccharide analysis

Current Monosaccharide analysis uses GC-MS, requires three days and ug for analysis.

New method allows 100 samples/day, with nanograms.

UPLC – QQQ MS

Dynamic MRM chromatogram of a fecal sample (P1A)

Sample GlcNAc GalNAc All Ara Fuc Gal GalA Glc GlcA Man Rha Xyl

P1A 5.42 3.09 0.22 0.46 5.18 19.95 0.11 9.65 33.28 1.01 0.07 0.40

(μg/mL)

4x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

Counts vs. Acquisition Time (min)

2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4

GlcAGal

Glu Fuc

GalA

3x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

Counts vs. Acquisition Time (min)2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4

Man

AllRha

GlcNAc

GalNAcXyl

Ara

Monosaccharide analysis in fecesQuantitation results of 22 fecal samples

(reducing monosaccharides)

Monosaccharides in feces show complementary (weaning) foods

HMO in feces increase until commensals are establishedGlycoproteins are effective pathogen block - Lactoferrin

Lactose

(Glyco)Lipids

Oligosaccharides (HMO)(Glyco)Proteins(Glyco)Peptides

Cells

RNADNA

MetabolitesMinerals Microbes

Micelles

Components of milk

• Glycosylation in proteins interact with the microbiome.

• Function as pathogen block.• New sources of prebiotic.

HMO in feces increase until commensals are establishedGlycoproteins are effective pathogen block - Lactoferrin

• Glycosylation on Lactoferrin varies during the lactation period.• Glycans are rich in fucose and sialic acids.

Mariana Barboza, Molec. Cell Proteom. 2012

Specific glycoforms bloc, distinct bacteria

Bart Weimar

Glycan Map of milk glycoproteins

Lactoferrin

IgG

IgA

alpha-1-antitrypsin

IgM

Huang et al. - Revision

• Systematic map of milk glycoproteins.• Extensive site heterogeneity.• Quantitate proteins and gycoforms.

Selection of Peptides for Quantitation (IgG Fc region)

IgG1

IgG2

IgG3

IgG4Peptide common in all the subclasses

Peptides unique for each subclass

N-glycosylation site

Protein identification and peptide quantification

Q Hong et al Anal Chem 2014

Quantitate protein (absolute)

Quantitate Glycopeptide (relative)

Normalize glycopeptide to protein abundance

IgG tryptic Glycopeptide - Quantitation MRM with UPLC QQQ MS

Each glycoform can be monitoredEach glycoform monitored in tryptic mixture.

Glycosylation site

Analytical Method for Proteins and Glycoproteins

• Multiple reaction monitoring – Mass Spectrometry

• Rapid throughput (15 min/run)• Sensitive – 10 uL milk• Absolute quantitation of 10-20

proteins with glycosylation.

0

0.1

0.2

0.3

1 2 3 4 5 6

Lyzo

(g/L

)

Month

Lysozyme

-1

1

3

5

7

1 2 3 4 5 6

LA (g

/L)

Month

Lactalbumin

0

0.5

1

1.5

1 2 3 4 5 6

IgA

(g/L

)

Month

IgA

0

0.05

0.1

0.15

1 2 3 4 5 6

IgM

(g/L

)

Month

IgM

0

0.1

0.2

0.3

1 2 3 4 5 6

IgG

(g/L

)

Month

IgG

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

0 2 4 6 8K Ca

sein

Abu

ndan

ce

Month

K Casein

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

0 2 4 6 8b Ca

sein

Abu

ndac

e

Month

b Casein USABrazilIndiaSouth AfricaPeruBangladesh

00.5

11.5

22.5

33.5

1 2 3 4 5 6

LF (g

/L)

Month

Lactoferrin

Abundance of Proteins among Countries – Month 1 to 6

Protein trends during lactation in countries

Trends in proteins are consistent across countries.

Protein behavior in severely stunted children

P = 0.0460

P = 0.0076

P = 0.0253

P = 0.0460

P = 0.0076

P = 0.0253

Trends in IgG glycosylation in different countries

IgG glycosylation Glycoforms change in the same pattern.IgG function and glycosylation.

Final Thoughts

• Get the best analytics possible• Diet changes total abundance, but not relative abundances of HMO.• HMO is everywhere. Both in the mother and in the infant. How does systemic HMO affect the mother?What role does systemic HMO have in infants?• HMO important in stunting and in infant health.• Protein/glycoproteins are yet unknown factors in infant development• Glycoproteins do not all decrease during lactation.Complicated behavior and differs with protein.• Glycoproteins function as pathogen block.

UC Davis Milk Bioactive Program

Autism

Preterm birthAnalytics

Clinical conditions

Environmental enteropathy

MicrobiomeInfection

ImmunityHost

Defense/infection

Gut Development

Brain Development

Microbiome DevelopmentObesity Compound

isolation/production

Bill and Melinda Gates Foundation