Document Detail

Proteomic analysis of kappa-casein micro-heterogeneity.
MedLine Citation:
PMID:  14997496     Owner:  NLM     Status:  MEDLINE    
The proteome of bovine milk is dominated by just six gene products that constitute approximately 95% of milk protein. Nonetheless, over 150 protein spots can be readily detected following two-dimensional electrophoresis of whole milk. Many of these represent isoforms of the major gene products produced through extensive post-translational modification. Peptide mass fingerprinting of in-gel tryptic digests (using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) in reflectron mode with alpha-cyano-4-hydroxycinnamic acid as the matrix) identified 10 forms of kappa-casein with isoelectric point (pI) values from 4.47 to 5.81, but could not distinguish between them. MALDI-TOF MS in linear mode, using sinapinic acid as the matrix, revealed a large tryptic peptide (mass > 5990 Da) derived from the C-terminus that contained all the known sites of genetic variance, phosphorylation and glycosylation. Two genetic variants present as singly or doubly phosphorylated forms could be distinguished using mass data alone. Glycoforms containing a single acidic tetrasaccharide were also identified. The differences in electrophoretic mobility of these isoforms were consistent with the addition of the acidic groups. While more extensively glycosylated forms were also observed, substantial loss of N-acetylneuraminic acid from the glycosyl group was evident in the MALDI spectra such that ions corresponding to the intact glycopeptide were not observed and assignment of the glycoforms was not possible. However, by analysing the pI shifts observed on the two-dimensional gels in conjunction with the MS data, the number of N-acetylneuraminic acid residues, and hence the glycoforms present, could be determined.
John W Holland; Hilton C Deeth; Paul F Alewood
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Proteomics     Volume:  4     ISSN:  1615-9853     ISO Abbreviation:  Proteomics     Publication Date:  2004 Mar 
Date Detail:
Created Date:  2004-03-03     Completed Date:  2004-10-18     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  101092707     Medline TA:  Proteomics     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  743-52     Citation Subset:  IM    
Institute for Molecular Bioscience, University of Queenslanf, Bribane, Australia.
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MeSH Terms
Amino Acid Sequence
Caseins / chemistry*
Electrophoresis, Gel, Two-Dimensional
Mass Spectrometry
Milk / chemistry*
Molecular Sequence Data
Peptides / chemistry
Protein Isoforms
Protein Processing, Post-Translational
Protein Structure, Tertiary
Proteomics / methods*
Serine Endopeptidases / chemistry
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Reg. No./Substance:
0/Caseins; 0/Peptides; 0/Protein Isoforms; 0/Proteome; EC 3.4.21.-/Serine Endopeptidases; EC endopeptidase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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