Abstract
The isotope-coded protein label (ICPL) technology has shown to be efficient for comparative quantification of proteins. A major aspect of this chemistry is the labeling of all free amino groups on the protein level. This allows for the application of protein fraction technologies such as 2D gel electrophoresis, liquid chromatography, or free-flow electrophoresis of the labeled protein sample under retention of the dynamic range in the sample. In addition, labeled free amino groups, which are sufficiently available in proteins, result in a high number of representative labeled peptides after protein digest. This is of high importance because at least five labeled peptide pairs are required for a reliable determination of regulation in proteins.
We used ICPL labeling for the analysis of a complex protein sample derived from the cell-wall proteome of Medicago truncatula. This plant belongs to the family of legumes that contribute about a third of the world’s protein requirements eaten by humans. Legumes are unique in their ability to fix atmospheric nitrogen through a symbiotic relationship with nitrogen-fixing bacteria Rhizobia. Thus, a comparative quantification study of extracted cell-wall proteins from M. truncatula is helpful for a better understanding of this particular plant-bacteria interaction.
The use of the newly developed ICPL triplex method will be shown, which allows for the comparative analysis of three proteomes. Furthermore, the data-handling aspects of multiplexed non-isobaric labeling was evaluated.