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Characterization of the differences in the host defense proteome of human and bovine milk 24 November 2011, Kasper Hettinga
Characterization of the differences in the host defense proteome of human and bovine milk
24 November 2011, Kasper Hettinga
Introduction to low-abundant milk proteins: proteomics
Host defense proteins in human and bovine milk
Future research plans
Topics
Introduction to low-abundant milk
proteins:
Proteomics
Cow’s milk contains: 3.5% protein 2.7% casein 0.7% serum proteins 0.1% MFGM protein
Image: Prof. Kees de Kruif
Fat
Which milk proteins exist?
Cow’s milk contains: 3.5% protein 2.7% casein 0.7% serum proteins 0.1% MFGM protein
Image: Prof. Kees de Kruif
Fat
Which milk proteins exist?
Cow’s milk contains: 3.5% protein 2.7% casein 0.7% serum proteins 0.1% MFGM protein
Image: Prof. Kees de Kruif
Fat
Which milk proteins exist?
Cow’s milk contains: 3.5% protein 2.7% casein 0.7% serum proteins 0.1% MFGM protein
Image: Prof. Kees de Kruif
Fat
Which milk proteins exist?
Cow’s milk contains: 3.5% protein 2.7% casein 0.7% serum proteins 0.1% MFGM protein
Image: Prof. Kees de Kruif
Fat
Which milk proteins are we interested in?
Milk
How to isolate these protein fractions?
Milk
Cream Milk plasma
Centrifuge
How to isolate these protein fractions?
Milk
Cream Milk plasma
Casein pellet Milk serum
Centrifuge
Ultracentrifuge
How to isolate these protein fractions?
Milk
Cream Milk plasma
Casein pellet Milk serum
Centrifuge
Ultracentrifuge
Wash Churn
Centrifuge Ultracentrifuge
Milk fat globule membrane proteins
How to isolate these protein fractions?
Milk
Cream Milk plasma
Casein pellet Milk serum
Centrifuge
Ultracentrifuge
Wash Churn
Centrifuge Ultracentrifuge
Milk fat globule membrane proteins
How to isolate these protein fractions?
LC-MS/MS Peptide & protein identification
MFGM
Trypsin
Peptide mixture of all proteins in 1 gel slice
16 chromatograms per sample
SDS-PAGE Milk
serum
Qualitative proteomics
Proteins solubilized in 4% SDS
Step 1: Washing away SDS etc.
Trypsin
Peptide mixture of all proteins
2 chromatograms per sample
FASP
LC-MS/MS Peptide & protein identification
Peptide & protein quantification
Optional: dimethyl labeling of peptides
Quantitative proteomics
Proteomics method – LC/MSMS-analysis
Result: chromatograms with thousands of peptides each Automatic peptide identification
High specificity of orbitrap-MSMS
Proteomics method – protein identification
Protein identification: database with amino acid sequences (>100.000 proteins): Fit jigsaw pieces (peptides) in a jigsaw puzzle
(protein) Check for false-positive identifications Known and “theoretical“ proteins (information from
the bovine genome) Protein quantification (label-free or dimethyl labeled)
Host defense
proteins in human and bovine milk
Experimental approach
Samples: 1 pooled human samples of 10 healthy nurses 3-12
months in lactation 1 pooled bovine sample of 30 healthy cows 2 weeks to 9
months in lactation Analyses: Qualitative comparison of all serum & MFGM proteins Quantitative comparison of host defense proteins
Human vs bovine – results (qualitative)
Qualitative (SDS-PAGE based) data: A total of 32 subsamples were analyzed using
LC/MSMS False-discovery rate: 0% (strict identification
criteria!)
268 human milk proteins and 269 bovine milk proteins
Human Bovine Overlap human-bovine
Total proteins 268 269 147 Serum proteins 222 192 105 MFGM proteins 234 232 118 Overlap serum-
MFGM 188 155
Human vs bovine – results (qualitative)
Human (n=268)
Human vs bovine
Cow (n=269)
Human (n=268)
Human vs bovine
Cow (n=269)
About 60% of all proteins are related to host defense or milk synthesis & secretion
Human vs bovine: milk synthesis proteins
Because human and bovine milk contain similar components, also the proteins associated with milk synthesis & secretion are expected to be similar
Central metabolic pathway: responsible for lactose and fat synthesis
Pathway Literature* Human Bovine Glucose to fatty acid 11 10 (9) 8 (8)
Lactose synthesis 2 4 (2) 2 (1)
Enzymes from the central metabolic pathway
*based on biopsies
Human vs bovine: milk synthesis proteins
Host defense proteins – introduction
Milk is important for the development of the immune system of the off-spring
Significant differences in the biological activity of infant formula and breast milk exist: related to difference in the host defense proteins?
System Human Bovine Overlap Complement 7 12 6
Immune response 9 10 8 Antibacterial 9 16 6
Immunoglobulins 15 9 5
Overview of qualitative differences in the host defense proteome
Host defense proteins – results
System Human Bovine Overlap Complement 7 12 6
Immune response 9 10 8 Antibacterial 9 16 6
Immunoglobulins 15 9 5
Overview of qualitative differences in the host defense proteome
Host defense proteins – results
It’s not only about which proteins, but also how much…
Quantitative differences in host defense proteins
Protein Human Bovine
CD14 44.4* 1.4
IgA 773* <1
Lactoferrin 1892.3* 52.7
Lysozyme 554.1* ND
Clusterin 25.5 <1
GLYCAM1 (PP3) <1 961.3
Lactoperoxidase 3.4 47.0*
Osteopontin 129.0 131.7
Milk serum
Host defense proteins – results
Quantitative differences in host defense proteins
Protein Human Bovine
CD14 44.4* 1.4
IgA 773* <1
Lactoferrin 1892.3* 52.7
Lysozyme 554.1* ND
Clusterin 25.5 <1
GLYCAM1 (PP3) <1 961.3
Lactoperoxidase 3.4 47.0*
Osteopontin 129.0 131.7
Milk serum
Defense system of mucosal secretions
Host defense proteins – results
Quantitative differences in host defense proteins
Protein Human Bovine
CD14 44.4* 1.4
IgA 773* <1
Lactoferrin 1892.3* 52.7
Lysozyme 554.1* ND
Clusterin 25.5 <1
GLYCAM1 (PP3) <1 961.3
Lactoperoxidase 3.4 47.0*
Osteopontin 129.0 131.7
Milk serum
Defense system of mucosal secretions
Host defense proteins – results
Human milk contains more of these proteins: baby is known to be deficient in IgA & CD14
Quantitative differences in host defense proteins
Protein Human Bovine
CD14 44.4* 1.4
IgA 773* <1
Lactoferrin 1892.3* 52.7
Lysozyme 554.1* ND
Clusterin 25.5 <1
GLYCAM1 (PP3) <1 961.3
Lactoperoxidase 3.4 47.0*
Osteopontin 129.0 131.7
Milk serum
Glycoproteins involved in apoptosis & cell repair
Defense system of mucosal secretions
Host defense proteins – results
Quantitative differences in host defense proteins
Protein Human Bovine
CD14 44.4* 1.4
IgA 773* <1
Lactoferrin 1892.3* 52.7
Lysozyme 554.1* ND
Clusterin 25.5 <1
GLYCAM1 (PP3) <1 961.3
Lactoperoxidase 3.4 47.0*
Osteopontin 129.0 131.7
Milk serum
Cow is plant eater? SCN-
from plant material
Glycoproteins involved in apoptosis & cell repair
Defense system of mucosal secretions
Host defense proteins – results
Quantitative differences in host defense proteins
Protein Human Bovine CD14 42.5 3.6* IgA 144* <1
Lactoferrin 2050.8 6.9* Lysozyme 196.3 <1
Clusterin 195.6 <1 GLYCAM1 (PP3) 3.3 300.2*
Mucins 41.4 46.3 Osteopontin 12.2 9.2
Milk MFGM
Amount of host defense proteins in MFGM is lower. Trends in differences between cow and human are the same Some specific MFGM proteins (e.g. mucins, clusterin)
Host defense proteins – results
Host defense proteins – conclusions
First direct quantitative comparison between bovine and human milk proteome
Many qualitative and quantitative differences in host defense proteins
Host defense proteins – conclusions
Higher concentration in human milk of proteins involved in the mucosal defense system: ● Newborn baby is deficient for two of these proteins ● Concentration difference between colostrum and
mature milk is much smaller for humans than cows ● Related to differences in maturation rate of the
immune system of babies compared to calves? Hettinga, K.A. et al (2011) The Host Defense Proteome of Human and Bovine Milk. PLoS One 6 (4): e19433
Future research plans
Future research plans
From proteomic
observations towards an understanding of biology
Future research plans
Milk synthesis & secretion: Understand variation in milk composition by studying
variation in the synthesis & secretion proteome
Some questions to be answered: ● Use the developed approach to understand the
synthesis & secretion of milk components (e.g. fat, protein)
● Compare proteome of MFGM to epithelial cells (either isolated from milk or biopsies)
● Link the results to transcriptome / genome data
Future research plans
Host defense proteins: Understanding of the biological implications of the
differences between human and bovine milk
Some questions to be answered: ● Variability of host defense proteins ● Biological activity of selected host defense proteins ● Health status of cow / mother / baby / calve ● Effect of processing and storage: stability
Thank you for your attention!
Acknowledgments Dairy science & technology group: Jing Lu, Hein van Valenberg, Lina Zhang, Toon van Hooijdonk, and many MSc students Laboratory of biochemistry: Jacques Vervoort, Sjef Boeren, Sacco de Vries Dutch milk genomics initiative
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