10/3/2016
1
Milk glycoproteomics: Preserving, enhancing, and
delivering bioactivity
Daniela Barile 1,3
Jaime Salcedo 1, Annabelle Le Parc 2, Adam Sun 2, Sercan Karav 1, Joshua Cohen 1, Juliana DeMoura Bell 1
1 Department of Food Science and Technology, University of California, Davis
2 Prolacta Bioscience, 757 Baldwin Park Blvd, City of Industry, CA
3Foods for Health Institute, University of California, Davis,
Milk as a model for functional ingredients
2
Infant
Adult
Bioactive components retained and amplified over 120 million years of evolution
Dr. Bruce GermanDirector FFHI
Dr. David MillsDairy Shields Chair
Dr. Carlito LebrillaDistinguished Professor
10/3/2016
2
Proteins
Peptides
Oligosaccharides
Glycolipids
Glycoproteins
How to get there: deconstructing milk bioactivity
Measure..measure..measure! Variety of monosaccharides
Numerous linkages and branching
Complex mixture
Issues of overlapping peaks
Intense sample purification required
O
O
CH2OH
OH
OH
OH
O
O
CH2OH
OH
OH
OH
Chiral CentersConnection sites
Retention time
ISOMERS
10/3/2016
3
Use multiple Analytical techniques• High performance anion exchange chromatography
• Quantification with standards
• Fraction collection
• nano‐LC‐Q‐ToF MS/MS• Full profile with fragmentation
• Include isomers separation
• MALDI‐ToF MS• Rapid profiling
• Identification of contaminants
Donor milk
• Increasing awareness of donor human milk benefits
• Expansion of human milk banks and industries collecting and processing
human milk
• Need to study the effect of thermal treatment on bioactive milk glycosylated
compounds
10/3/2016
4
Previous findings from the literatureYear Author (et al.) Target Compunds # of samples Heat treatment Results
1996 TroyanoMonosaccharides & aminosugars
(Bovine milk)4 UHT Slight increase
2008 E. Bertino Oligosaccharides 10 Holder Pasteurization No Significant Change
2011 J. Ewaschuck
Lactose 12‐15 Holder Pasteurization No Significant Change
IgA 1‐36 Holder Pasteurization 0%‐48% Reduction
IgG 9 Holder Pasteurization 34% Reduction
Lactoferrin 1‐22 Holder Pasteurization 57%‐80% Reduction
Lysozome Activity 1‐36 Holder Pasteurization 0%‐60% Reduction
2013 C. Marx Oligosaccharides 5 Holder Pasteurization No Significant Change
2015 J. Ding Lactose 3 Retort Processing No Significant Change
2016 C. Peila
Lactose 10 Holder Pasteurization No Significant Change
IgA 1‐36 Holder Pasteurization 20%‐62% Reduction
IgG 1‐36 Holder Pasteurization 23%‐100% Reduction
Lactoferrin 1‐22 Holder Pasteurization 35%‐90% Reduction
…what about other thermal treatments?
Is there detectable/quantifiable difference in milk bioactive components due to various heat treatments and/or
storage conditions?
10/3/2016
5
Study design
Heat Treatment #2high temp‐short time(HTST) 72 °C 15 sec
Heat Treatment #3extended shelf life (ESL) 127 °C 5 sec
Heat Treatment #4ultra‐high temperature
(UHT) 140 °C 6 sec
Heat Treatment #1Holder Pasterur.63 oC 30min
Filling under sterile conditions
Non‐treated milk (baseline control)
20L of pooled human milk from 5 donors
Abundance and number of structures for:
Human Milk OligosaccharidesProtein N‐glycosylation
GlycolipidsNano LC Chip Q ToFBarile Lab
Sterile product outlet automatic filling control
(MicroThermics)
UHT/HTST hybrid continuous pasteurizer
(MicroThermics)
Donor milk from Prolacta Bioscience Thawing & pooling
Investigate thermal effect on milk glycosylated components
triplicates
Mat & Methodsand Results
HMO
N‐GLYCANS
GLYCOLIPIDS
Dr. Adam Sun
Dr. Salcedo
Dr. LeParc
10/3/2016
6
Human Milk Oligosaccharides
D-Galactose
D-Glucose
N-acetyl-glucosamine
L-Fucose
Sialic acid
5 building blocks
Many possible linkages
TERMI
NAL
POSI
TI
ON
Human Milk Oligosaccharides Functions
Development of nervous system
Selective prebiotics
Decrease hypersensitivity
reactionsGut barrier function
Antibacterial and antiviral
Immune system activation
10/3/2016
7
HMO isolation and analysis100 L milk + 100 L H2O
Fat removal(Centrifugation 4000rpm,30min,4°C)
Lower layer(lipids)
Folch extraction (CHCl3/MeOH 2:1)
Pellet Upper layer(lipids)Supernatant
(Proteins, HMOs…)
Upper layer
(proteins, HMOs…)
Protein precipitation: EtOH (‐20C, 1h) + centrifugation
Pellet(proteins) Supernatant
(HMOs, salts….)
Reduction and solid phase extraction
Neutral Fucosylated
Neutral
Sialylated Fucosylated
Sialylated
HMO profile of the raw milk (control)
14
49%
34%
10%
5%
OS Relative Abundance (%) by type
65 individual structures monitored for abundanceIn the Control and in the 4 heat‐treated samples
………………….
10/3/2016
8
Mat & Methods
and Results
HMO
N‐GLYCANS
GLYCOLIPIDS
Dr. Karav
18
N‐glycans & health
Protein structure and function (antioxidant, antibacterial and antiviral activities, binding of iron, and immunomodulation)
Protein structure and function
Selective prebioticactivity
Inhibition of pathogens and immunomodulation
10/3/2016
9
N‐glycan release: classical method
19
Lipid Removal
Lipids
Proteins, free oligosaccharides
pellet
Centrifuge30min 4 0C
Protein Precipitation
1V Milk + 4V cold ethanol
Enzymatic Release
Released N‐glycanspurification
Characterization bymass spectrometry
Ethanol precipitation+PGC
PNGase F
90 C
Sample heating for protein denaturation is required for
PNGaseF
(it would mask the effect of thermal treatment)
N‐glycan release optimization
Initial discovery byDr. Daniel Garrido, (Dr. Mills’ Lab)
• Recombinant enzyme Endo BI‐1• Isolated from B. longum subsp. infantis• Active on native glycoproteins • No need for protein denaturation• Simulates N‐glycans accessibility in the gut of healthy breast‐fed infants
OptimizationBy Dr. Karav
(Mills & Barile Lab)
(Endo BI‐1)
10/3/2016
10
Modified protocol for N‐glycan Release
Lipid Removal
Lipids
Raw Milk
Proteins, free oligosaccharides
Caseins
Centrifuge30min 4 0C
Protein Precipitation
HMO Removal
1V Milk + 4V cold ethanol
10 kDAmembrane separates proteins from HMOs
HMO
Proteins
Enzymatic Release: EndoBI‐1Glycan CollectionCharacterization byNano LC Chip QTOF
Ethanol precipitation+PGC
37 ᴼC
Glycan Diversity (Number/type of structures)
Relative Abundance of all structures
N‐glycans analysis
Glycosidase
10/3/2016
11
N‐glycans abundance: comparing control, HT1, HT2
HT 1: 63 °C 30 min HT 2: 72 °C 15 sec
N‐glycans abundance:comparing control, HT3, HT4
HT 3: 127 °C 15 min HT 4: 140 °C 6 sec
10/3/2016
12
Mat & Methodsand Results
HMO
N‐GLYCANS
GLYCOLIPIDSDr. Salcedo
Glycolipids
29
R
OH OH
NH
R
O
-O-Glucose -Galactose -Sia
Ceramide
Fatty acid(18-24 C)
Sphingoid base (16-20 C)
Oligosaccharide chain
Glycolipids: Glycosphingolipids containing at least one Sia moiety in the structure
> 100 compounds
10/3/2016
13
Glycolipids
Pathogens
inhibition
Decoy
Prevent infection
Prebioticactivity
bifidobacteria
Activate T cellsand Th1-Th2lymphocytes
Anti-inflammatoryeffects
Immuneresponse
Mielin stability
Nervous tissueregeneration
Neuronal development
Glycolipids and health
30
Crude glycolipid extract
Clean up
C‐8 cartridges
N‐omega‐ CD3‐octadecanoyl GM3
(internal standard)
1.5 mL sample
Lipid extract Glycolipid extract
Lipid extract
+
MetOH:CHCl3:H2O(8:4:2)
+Glycolipid extraction
UPLC‐QQQ
Lebrilla’s lab
Glycolipid extraction & purification
MetOH:H2O(2:1)
Glycolipid extract
10/3/2016
14
Total HMO abundance Glycolipids are stable Results
N‐glycans
Translation: Bovine Milk Oligosaccharides
Monitor oligosaccharides enrichment at the pilot scale from whey permeate
Dr. JulianaDeMoura Bell
Josh Cohen(see Poster)
10/3/2016
15
What happens to oligosaccharides
during spray dry & storage?
As an emerging functional ingredient,
there is a need to understand the
chemical reactivity of this ingredient
within the dairy matrix
http://www.snexportgroup.com/
structural modifications
?
REDUCEDSOLUBILITY
?
Bioavailability?
Degradation products?
?
10/3/2016
16
http://www.gea.com/
PROBLEMATIC QUANTIFICATION
Good resolution& QUANTIFICATION
Maillard reaction
• Leads to the decrease in nutritional value of proteins and formation of brown compounds in milk
• Loss of essential amino acids
• Amadori Products can be measured by a number of chromatographic and enzymatic techniques (assess the reaction stage)
Reaction between reducing sugars and amino acids
Accelerated during heat treatment.
Continue to develop during long storage (RT) Louis-CamilleMaillard, 1916
10/3/2016
17
• Evaluate the formation of Amadori Products involving BMO
• Assess the nutritional impact of the reaction in infant formulaenriched with BMO
Prof. Vincenzo Fogliano
• Chair of Food Quality Design• Former President of the International
Maillard Reaction Society (IMARS)
O
OH
O
NH2acrylamide
OO
glyoxal
O
O
methylglyoxal
O
H
HO
H
HO
H
OHOHH
H
OH
H2NH2N O
OH
lysine
aldosesugar
+amino
compoundN-subsituted glycosylamine + H2O
Amadori rearrangement
1-amino-1deoxy-2-ketose
- 3 H2O
Schiff base of HMFor furfural
- amino compound+ H2O
-2 H2O
reductones
+ -amino acidsStrecker degradation
-2H +2H
CO2
dehydroreductonesHMF or furfural
+ amino compound
aldimines
with or without amino compound
aldols and N-freepolymers
+ amino compoundaldimines orketimines
aldehyde
+ amino compound
aldimines
+
fissionproducts(acetol,
pyruvaldehyde,diacetyl etc)
O
2-methylbutanal
MelanoidinsBrown nitrogenous polymers and copolymersstyrene
O
HO
acetic acid
N
N
pyrazine
NH
pyrroline
Initial stage
Intermediate stage
Advanced stage
HMF
O
O
2,3-Butanedione
Maillard reaction: Hodge schema
Hodge; J.Agric Food Chem, 1953
BMO ?
10/3/2016
18
BMO Degradation and Amadori Products formationBMO Degradation and Amadori Products formation
Air forced circulating oven
Exactive OrbitrapHRMS system
Commercial Infant Formula
Same infant Formula Enriched with BMO
( UC‐Davis MPL)
BMO Model System
( Pure Standards + amino acids )
Oligosaccharidesaminoacids &
Amadori Products
Thermal treatment Quantification
Whirlpool, 900W M420 Double Emission System)
Personal Compound Database and Library
Many fucosylated oligosaccharides (albeit in low abundance)
2016
Exp. spectra comparison with spectral library produces a match score allowing rapid & confident compound identification
Dr. Otter
10/3/2016
19
Assembling more tools to achieve the full analytical characterization of bioactive glycosylated compounds in human milk and dairy products…
quoteaddicts.com
Disclosure: I am co-founder of a start-up company (Evolve BioSystems) that studies probiotics and prebiotics
The Peter J. Shields Endowed Chair in Dairy Food ScienceThe Peter J. Shields Endowed Chair in Dairy Food Science
10/3/2016
20
Thank you for your attention
Speed Bump by Dave Coverly