Results and discussion

Gel based proteomics

Proteomic analysis of protein changes in milk products during processing and storage

Thao T. Lea,b,c, John W. Hollandb, Hilton C. Deetha and Lotte Bach Larsenc aSchool of Agriculture and Food Sciences, The University of Queensland, Australia bInstitute for Molecular Bioscience, The University of Queensland, Australia cDepartment of Food Science, Aarhus University, Denmark. Contact: Thao.ThiThuLe@agrsci.dk

AARHUS UNIVERSITY

Acknowledgments

We thank Alun Jones and Hanne Søn-dergård Møller from the IMB mass spec-trometry facility, The University of Australia and Department of Food Science, Aarhus University respectively for their expertise.

Table 1. Lactosylated sites of whey proteins identified in MPC80 and WPI. Tryptic digests of milk proteins were analy-

sed by an ion trap Esquire6000 coupled to the capillary LC.

Figure 4. Product-ion of tandem MS of the triply charged ion at m/z 638.4 in full scan (above) and zoomed in (below) spectra.

Figure 3. MALDI-TOF spectra of intact α-La and in-gel tryptic digests extracted from 2-DE gel spots of MPC80 sample sto-

red at 250C, 84% RH for 4 weeks.

Figure 1. General process of proteomic analysis of milk

samples.

Gel free proteomics

Conclusion

- Substantial changes were observed in milk powders stored at high temperature and/or high humidity.

- 2-DE is a good separation technique for protein modifications in milk samples experienced minor changes (e.g., mild storage condition).

- LC combined with MS can be used as a direct tool for quantification of lactosylati-on in milk products (e.g., multiple reaction monitoring approaches).

Introduction

- Heat-treated milk products can undergo chemical changes (e.g., lactosylation) during processing and storage which can cause deterioration of its functional pro-perties and nutritional quality.

- Proteomics is a unique tool to characterise post-translational modifications in milk pro-teins (e.g., glycosylation, phosphorylation, disulphide bond formation and proteolysis) (Gagnaire et al., 2009).

- We have applied proteomic techniques to analyse chemical changes occurring in milk powders during processing and storage.

Objectives

- To use two-dimensional gel electropho-resis (2-DE) to observe modifications in the protein profile produced by chemical changes during storage of milk powders.

- To use mass spectrometry (MS) in com-bination with liquid chromatography to separate and characterize the specific changes occurring in milk proteins.

Materials and methods

Milk powders (milk protein concentrate and whey protein isolate containing 80% of protein labelled as MPC80 and WPI respectively) were stored at a range of tem-peratures (25-400C) and relative humidities (RH) (44-84%) up to 4 weeks. The samples were analysed by 2-DE, MALDI-TOF-MS and ESI-LC-MS/MS (Figure 1).

Figure 2. Reducing 2-DE of MPC80 stored at different tem-peratures and humidities for 4 weeks. Major milk proteins are labeled in control sample. The boxes show regions where high-molecular-weight protein complexes and vertical spot

stacking of whey proteins can be observed.

- Formation of high-molecular-weight protein complexes are temperature- and humidity-dependent. AlphaS1-casein (αS1-CN), alphaS2-casein (αS2-CN), beta-casein (β-CN), kappa-casein (κ-CN) and beta-lactoglobulin (β-Lg) are present in the cross-linked complexes (results not shown).

- The appearance of vertical spot stacking in α-La and β-Lg is due to the presence of lactose adducts

- For the intact protein: each spot in the stack shows an increment of 324 Da which is the mass of a lactose adduct.

- For the tryptic digest: the asterisk * labels the peptides modified by lactose. This was observed in spots 2, 3 and 4.

- 8 and 9 lactosylated sites out of 15 and 12 lysine positions of β-Lg and α-La respec-tively were characterised in stored MPC80 and WPI.

- The neutral losses including the loss of galactose [M-162] and the formation of furylium ion [M-216] (loss of 3H2O) are unique fragmentations of glycated pepti-des.

- These ions can be used as specific transi-tions in multiple reaction monitoring met-hods in order to select the best candidates for lactosylation quantification of milk proteins (Le et al., 2013).

References

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