Following one of these preparations, the selectively labeled protein thiols can be assessed by a range of analytical procedures based on the nature of the thiol label that has been employed (Figure 2c). Typically, the labels attached

to the cysteine residues are biotinylated, fluorescently conjugated, or isotopically modified derivatives of the thiol alkylating reagents NEM or iodoacetaminde (IAM). Such labeling procedures allow for separation of the proteins by gel electrophoresis followed by the identification of the labeled protein by peptide mass fingerprinting, or by the separation and identification of the labeled peptides by liquid chromatography–mass spectrometry (LC–MS). The details of these labeling methods are first discussed below, followed later by a comparison of the various types of thiol-reactive probe molecule that can be used and procedures

that can be employed to separate and identify the labeled SAHA HDAC chemical structure proteins and peptides. A simple but limited strategy for the identification of modified protein thiols is to label all unmodified protein thiols with detectable thiol-reactive probes (Figure 2b, Selleckchem INK128 top) [28, 29 and 30]. Then control labeled protein samples can be separated by electrophoresis, or derived peptides are separated by LC–MS, and compared with related samples prepared under stressed or oxidant-treated conditions. Probe signal loss between conditions is indicative of both reversibly and irreversibly modified protein thiols (Figure 3a). However, the reliance on measuring signal loss, instead of signal increase over baseline, is a significant limitation to the sensitivity of this approach since most intracellular protein thiols are maintained in a reduced state by the glutathione and thioredoxin systems.

Consequently, this strategy is best suited for determining changes due to high concentrations of oxidants or in simple protein samples. The signal loss method can in principle be adapted to detect only irreversible protein thiol modifications by the treatment of samples with a thiol reductant, such as tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) before labeling. In this case any RVX-208 signal loss would be attributable to irreversibly oxidized thiols. A more widely used strategy, and one that is generally the most useful for the detection of reversibly modified protein thiols, blocks all unmodified thiols with a general thiol reagent such as NEM. This is followed by the selective reduction and labeling of all reversibly modified cysteine residues with a thiol probe. All redox-sensitive cysteine residues will be labeled and screened for by this procedure, regardless of the nature of the reversible modification (Figure 3b and c). This is advantageous when the conditions being compared involve a range of reversible modifications, of which the combination and proportion are unknown.