Exploring Milk Components by Glycoproteomics and Peptidomics
Journal
Proteomics Applied to Foods
Series/Report No.
Methods and Protocols in Food Science
Part Of
Proteomics Applied to Foods
Start Page
51
End Page
72
ISBN (of the container)
9781071640753
9781071640746
9781071640777
DOI (of the container)
10.1007/978-1-0716-4075-3
ISBN
9781071640753
9781071640746
Date Issued
2024-10-03
Author(s)
Abstract
Glycoproteins, naturally occurring peptides, and oligosaccharides in milk play unique roles in modulating human health. The application of omics techniques can attain comprehensive structural and compositional characterization of these components of milk. Here we describe protocols for characterizing milk oligosaccharides, naturally occurring peptides, proteomes, and protein glycosylations using liquid chromatography-tandem mass spectrometry (LC-MS/MS), with a focus on the sample preparation procedures. Free oligosaccharides and naturally occurring peptides are purified by removing other milk components through a series of extraction steps, including skimming, Folch extraction, protein precipitation, and solid-phase extraction. The preparation for proteomics and O- and N-linked glycan analyses begins with protein denaturation, reduction, and alkylation by employing a filter-aided approach. The denatured and alkylated protein samples are then divided into two portions. One is used for O- and N-glycan release by β-elimination and PNGase F treatment, respectively, and the other undergoes protein digestion to obtain tryptic peptides. In subsequent LC-MS/MS analysis, chromatographic separation is achieved using a nano-flow on a porous graphitized carbon column for glycan samples (including free oligosaccharide and released O-/N-glycans), while peptide samples are separated on a C18 column. These procedures can be applied to milk from different mammals as well as to dairy streams generated by a range of processing techniques.
SDGs
Type
book part