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The advent of mass spectrometry–based tagging methods, in particular amine-specific labeling reagents, has permitted relative expression measurements of large protein sets with a high degree of accuracy and reproducibility. The isobaric nature of the tags allows the protein samples to be pooled after labeling without increasing the complexity of the MS analysis. Identical peptides labeled with the different N-terminal reagents exhibit the same parent ion in MS. Upon MS/MS fragmention of the parent ion, unique signature ions are generated, which indicate the source sample of each peptide and therefore the peptide’s relative abundance among the samples determined.
As tagging technology has become established and gained acceptance in relative protein expression analysis, there is a need to expand the scope of this technique to enable a higher degree of multiplexing. Described herein is a new set of reagents that doubles the number of states that can be compared from four to eight using the same robust N-hydroxysuccinimide chemistry and easy-to-use protocols as the original 4-plex amine-specific tags. Examples of the use of this reagent to quantitate eight states simultaneously will be shown and evaluated for label efficiency, fragmentation efficiency, and precision and accuracy of quantitation.