Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy

Proteomics
John W HollandPaul F Alewood

Abstract

Visualisation of multiple isoforms of kappa-casein on 2-D gels is restricted by the abundant alpha- and beta-caseins that not only limit gel loading but also migrate to similar regions as the more acidic kappa-casein isoforms. To overcome this problem, we took advantage of the absence of cysteine residues in alpha(S1)- and beta-casein by devising an affinity enrichment procedure based on reversible biotinylation of cysteine residues. Affinity capture of cysteine-containing proteins on avidin allowed the removal of the vast majority of alpha(S1)- and beta-casein, and on subsequent 2-D gel analysis 16 gel spots were identified as kappa-casein by PMF. Further analysis of the C-terminal tryptic peptide along with structural predictions based on mobility on the 2-D gel allowed us to assign identities to each spot in terms of genetic variant (A or B), phosphorylation status (1, 2 or 3) and glycosylation status (from 0 to 6). Eight isoforms of the A and B variants with the same PTMs were observed. When the casein fraction of milk from a single cow, homozygous for the B variant of kappa-casein, was used as the starting material, 17 isoforms from 13 gel spots were characterised. Analysis of isoforms of low abundance proved challenging d...Continue Reading

References

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Citations

Sep 14, 2011·Journal of Agricultural and Food Chemistry·Oswaldo Hernández-HernándezF Javier Moreno
Feb 19, 2013·Journal of Agricultural and Food Chemistry·Therese Uniacke-LoweDaniel M Mulvihill
Sep 11, 2008·Genetics·Anna-Maria TyriseväMatti Ojala
Jan 9, 2014·Glycobiology·Noelle O'RiordanRita M Hickey
May 30, 2012·Journal of Proteomics·Paola RoncadaLuigi Bonizzi
Oct 2, 2009·Journal of Proteomics·Begoña CasadoMartin Kussmann
Jun 17, 2009·Journal of Proteomics·Albert CasanovasMiquel Llobera
Apr 19, 2011·Mass Spectrometry Reviews·Priscille GironJean-Charles Sanchez
Oct 12, 2015·Journal of Dairy Science·Ruben de VriesKasper Hettinga
Feb 18, 2014·Biochemical and Biophysical Research Communications·Míriam Badia-VillanuevaM Dolores López-Tejero
Sep 18, 2014·Animal : an International Journal of Animal Bioscience·S S LiJ X Liu
Sep 25, 2020·Food Science of Animal Resources·Iqra YasminAdnan Khaliq
May 7, 2010·Journal of Proteome Research·Angelo D'AlessandroLello Zolla
Jun 2, 2021·Critical Reviews in Food Science and Nutrition·Sara BenedéElena Molina
May 28, 2010·Journal of Agricultural and Food Chemistry·François Chevalier, Alan L Kelly
Feb 17, 2011·Journal of Agricultural and Food Chemistry·John W HollandPaul F Alewood

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