Vicinal dithiols, which are likely to form intraprotein disulfides because of their proximity, can be identified on the basis of a selective labeling and reduction strategy. Protein dithiols in reduced protein samples can be selectively blocked with the dithiol specific reagent phenylarsine oxide (PAO) and then all other thiols alkylated with

NEM. Subsequently, PAO-blocked dithiols are selectively reduced using the PAO-specific reducing agent 2,3-dimercaptopropanesulfonic acid (DMPS) and labeled with an alkylating probe [19, 46 and 47]. Identification of novel proteins that undergo inter-protein disulfide formation is also possible using diagonal electrophoresis [48]. Protein samples are first resolved by non-reducing SDS-PAGE so that all thiol modifications remain intact. Then samples are resolved in the second dimension with DTT incorporated into the running medium. By incorporating the reduction ALK inhibitor clinical trial step at this point, proteins involved in inter-protein disulfide linkages will migrate off the diagonal and can be subsequently identified by peptide mass fingerprinting or with an antibody on a western blot if candidate proteins are suspected. The reliance of this technique on electrophoresis limits the potential resolving power for complex protein mixtures. This lack of sensitivity can be addressed to some extent if a thiol specific fluorescent probe

is incorporated during the reduction step. Although this would focus on the cysteine residues, Ipilimumab in vivo in this case other thiol modifications in addition to inter-protein disulfides would also be labeled. As both the glutathione and thioredoxin systems are critical for the maintenance of protein thiol redox homeostasis, techniques Montelukast Sodium have been developed to identify the protein targets of these interactions.

Lind et al. used a mutant glutaredoxin from E. coli to selectively reduce glutathionylated proteins following the general scheme described in Figure 3b [ 49•]. Although this strategy may identify constitutively glutathionylated proteins it is unclear if the mutant glutaredoxin is capable of reducing all glutathionylated proteins. Sensitive strategies for the identification of thioredoxin-conjugated proteins have relied on the blocking of unmodified thiols, followed by the treatment of oxidized thiols ± thioredoxin and blocking of thioredoxin-reduced thiols. Finally, oxidized thiols not affected by thioredoxin treatment are reduced and labeled resulting in a signal [ 50•]. Decreased signal probe intensity in thioredoxin treated samples is indicative of a target cysteine residue. Recently, Benhar and colleagues used a combined strategy of selective reduction of protein S-nitrosothiols and thioredoxin conjugation to specifically determine S-nitrosated targets of thioredoxin action [ 51]. Using stable isotope labeling by amino acids in cell culture (SILAC), entire proteomes can be differentially labeled with light or heavy lysine.