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A non-isobaric label chemistry is introduced that allows the triplexed quantification of proteomic samples based on the isotope-coded protein-labeling technology (ICPL). Amino groups of intact proteins were derivatized with three isotopically different nicotinoyl reagents (L: 12C6H4; M: 12C6D4; and H: 13C6H4) prior to protein separation and digestion. Here, we describe different approaches in respect of MS instruments and protein separation.
Protein samples with different complexity were labeled with ICPL. For one sample, the proteins were digested, separated by CAP LC, and spotted on disposable targets for subsequent MALDI measurements. The other sample was separated on a 1D gel, excised, digested, and supplied to LC-MS/MS using a Qq orthogonal time-of-flight instrument.
Excellent results were obtained from the LC-MALDI and the Qq TOF approach with the three protein samples labeled differentially and measured together. The multiplexed labeling allows us to reduce the experimental effort when proteomics experiments are run as replicates and when more than two different states are to be compared. A unique property of ICPL compared to other label chemistries is its compatibility with protein pre-fractionation, as the labeling reaction works on the undigested protein.