24 November 2011, Kasper Hettinga · defense proteome of human and bovine milk 24 November 2011,...

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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

Which milk proteins exist?

Which milk proteins are we interested in?

How to isolate these protein fractions?

Cream Milk plasma

Centrifuge

Cream Milk plasma

Casein pellet Milk serum

Centrifuge

Ultracentrifuge

Cream Milk plasma

Centrifuge

Ultracentrifuge

Wash Churn

Centrifuge Ultracentrifuge

Milk fat globule membrane proteins

Cream Milk plasma

Centrifuge

Ultracentrifuge

Wash Churn

Centrifuge Ultracentrifuge

Milk fat globule membrane proteins

LC-MS/MS Peptide & protein identification

Trypsin

Peptide mixture of all proteins in 1 gel slice

16 chromatograms per sample

SDS-PAGE Milk

Qualitative proteomics

Proteins solubilized in 4% SDS

Step 1: Washing away SDS etc.

Trypsin

Peptide mixture of all proteins

2 chromatograms per sample

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

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

CD14 44.4* 1.4

IgA 773* <1

Lysozyme 554.1* ND

Clusterin 25.5 <1

GLYCAM1 (PP3) <1 961.3

Milk serum

Defense system of mucosal secretions

CD14 44.4* 1.4

IgA 773* <1

Lysozyme 554.1* ND

Clusterin 25.5 <1

GLYCAM1 (PP3) <1 961.3

Milk serum

Human milk contains more of these proteins: baby is known to be deficient in IgA & CD14

CD14 44.4* 1.4

IgA 773* <1

Lysozyme 554.1* ND

Clusterin 25.5 <1

GLYCAM1 (PP3) <1 961.3

Milk serum

Glycoproteins involved in apoptosis & cell repair

CD14 44.4* 1.4

IgA 773* <1

Lysozyme 554.1* ND

Clusterin 25.5 <1

GLYCAM1 (PP3) <1 961.3

Milk serum

Cow is plant eater? SCN-

from plant material

Glycoproteins involved in apoptosis & cell repair

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 – 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

From proteomic

observations towards an understanding of biology

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

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

kasper.hettinga@wur.nl

24 November 2011, Kasper Hettinga · defense proteome of human and bovine milk 24 November 2011,...

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