Purification and identification of water-soluble phosphopeptides from cheese using Fe(III) affinity chromatography and mass spectrometry.
Abstract
Water-soluble phosphopeptides from cheese were isolated using immobilized metal affinity chromatography (IMAC). Phosphopeptides from aqueous cheese extracts were completely retained on iminodiacetic acid (IDA) Sepharose equilibrated with FeCl3 and subsequently eluted with ammonium dihydrogen phosphate. Peptides in the eluate from the IMAC-Fe(III) column were identified using reversed phase liquid chromatography-electronic spray identification-tandem mass spectrometry. Phosphopeptides from two different cheeses were analyzed using the described method: a 10-month-old semihard Herrgard cheese made with mesophilic starter and a 24-month-old Parmigiano Reggiano cheese made with thermophilic starter. Elution of the IMAC-Fe(III) column with a gradient of ammonium dihydrogen phosphate resulted in three distinct peaks for Herrgard cheese corresponding to peptides carrying one, two, and four phosphorylated serine residues, respectively. Sixty-five different phosphopeptides were identified from the Herrgard, whereas only 9 from the Parmigiano Reggiano.
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These phosphopeptides share a common feature: the cleavage site in 28th position; plasmin can cleave the peptide bond at Lys28-Lys29 of β-casein, but thermophilic starter bacteria may play an important role.
“These phosphopeptides share a common feature: the cleavage site in 28th position; plasmin can cleave the peptide bond at Lys28-Lys29 of β-casein (Lund and Ardö, 2004), but thermophilic starter bacteria may play an important role, as well.”
From The Interrelationship Between Microbiota and Peptides During Ripening as a Driver for Parmigiano Reggiano Cheese Quality (2020) · cites this paper as Lund and Ardö, 2004
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- Authors
- Lund, M., & Ardö, Y.
- Affiliations
- Department of Food Science, Dairy Technology, Centre for Advanced Food Studies, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
- Corresponding author
- Ardö, Ylva
- Venue
- Journal of Agricultural and Food Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2004-09-18
- Volume
- 52
- Issue
- 21
- Pages
- 6616-6622
- ISSN
- 0021-8561 (ISSN-L)
- 1520-5118
- OpenAlex
- W1963608004
- Cited by
- 41
- References
- 20
- Citations by year
- 2025: 1 · 2021: 2 · 2020: 3 · 2017: 3 · 2016: 3 · 2013: 5 · 2012: 5
- FWCI
- 1.11
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Agricultural and Food Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (submittedVersion)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Agricultural and Biological Sciences › Food Science › Food and Agricultural Sciences
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Hydrolysis and Bioactive Peptides
- Keywords
- phosphopeptides, cheese, immobilized metal affinity chromatography, Parmigiano-Reggiano cheese, serine phosphorylation, starter cultures
- MeSH terms
- Cheese, Chromatography, Affinity, Ferric Compounds, Phosphopeptides, Solubility, Mass Spectrometry, Water
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