ECAT Reagents

Add 100 pg of the control protein solution to one vial of dissolved normal isotope ICAT reagent. Mix to dissolve. Add 100 pg of the test protein solution to one vial of dissolved heavy isotope ICAT reagent. Mix to dissolve. 

Combine the test sample with the control sample in a single vial. Mix well. 

Prepare a solution of TCPK-trypsin in 50 mM ammonium bicarbonate, pH 8.0, at a concentration of 100 ng/pl. Add 10 pi of the trypsin solution to every 10 pg of combined, labeled protein solution from Step 6. Incubate at 37°C for 12-16 hours or overnight with mixing. 

The O-ECAT reagent is a superior alternative to the use of 2,4-dinitrophenylhydrazine (DNPH Chapter 1, Section 1.1) in the study of protein oxidation. DNPH modification produces detectable complexes, but it does not provide information as to what amino acids are involved. O-ECAT modifies carbonyl end products of protein oxidation and in addition, it can provide exact information as to the amino acids that were oxidized. Mass spec analysis of modified proteins performed after proteolysis gives the exact amino acid sequences including the sites of O-ECAT reagent modification. The same antibody that is specific for the metal chelate portion of the standard ECAT reagent also can be used to capture and detect the O-ECAT 

ECAT mass tag linked to protein via thioether bond 

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Figure 16.8 Reaction of the ECAT reagent with a cysteine-containing protein results in the formation of a stable thioether bond.

Figure 16.9 The O-ECAT reagent structure contains a DOTA chelating group and a terminal aminoxy group for coupling to aldehyde and ketone sites of oxidation within biological molecules.

The use of mass tagging reagents to analyze proteomic data has greatly improved the ability to compare samples for protein expression differences. However, a major limitation of the ICAT procedure (Section 1, this chapter) is that it can only compare two samples simultaneously, usually a test and a control. Even with the ECAT design (Section 2) using multiple lanthanide metals to make a series of different mass tag signatures, it is difficult to extend the... 

The protocol for using isobaric tags differs from that described previously for the ICAT or ECAT type reagents. In the following method, the proteins are denatured and the disulfides reduced and then alkylated to block them permanently. This eliminates disulfide re-association and also prevents the isobaric tags from forming thioester modification with cysteine thiols. Next, the proteins are digested with trypsin and then modified with an isobaric tag. Each sample is labeled with a different isobaric compound so that the samples can be differentiated upon MS/MS analysis. 

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