One subset of protein thiols that may be of particular interest are those in mitochondria, as these thiols are most likely to be involved in antioxidant defense against ROS production by the mitochondrial respiratory chain as well as in redox signaling. Additionally, the protein thiol content in mitochondria is high and the high local pH (∼8) makes surface thiols within this compartment more reactive [23]. Generally the study of mitochondrial protein thiols is conducted using

isolated mitochondria; however, the use of mitochondria targeted compounds, such as MitoSNO [24] and (4-iodobutyl)triphenylphosphonium [25 and 26] enable the selective modification of mitochondrial protein thiols within

more complex systems, such as cells and whole organisms. Most of the approaches used for the study of mitochondrial protein thiols can be applied to the investigation of other sub-cellular compartments PLX3397 or of the entire cell (Figure 2a). Here we discuss the Selleck Alpelisib general methods available for the labeling of protein thiol modifications by selective probes and the separation and identification of the proteins containing particular cysteine redox modifications. In all cases the strategies are given in general terms and readers are referred to references for technical details from representative studies. When discussing these methods an effort has been made to mention techniques used to identify endogenously produced modifications or in vivo redox status because these approaches tend to be the most sensitive and relevant for wider application. Many thiol modifications on cysteine residues are Carnitine palmitoyltransferase II relatively labile and thiols themselves

are prone to artifactual modification during protein isolation and labeling. Therefore an essential prerequisite for reliable screening for protein thiol modifications in biological samples is the efficient trapping of the native redox state of the thiol proteome [27]. This is generally done using a reactive thiol alkylating reagent such as N-ethyl maleimide (NEM) to block all free thiols, a step which is sometimes preceded by treatment with strong acid to protonate the thiols and render them less reactive [27]. There are three general approaches that are used for the labeling of cysteine residues within samples for most redox proteomic studies (Figure 2b). Either unmodified protein thiols are alkylated with a thiol specific probe that contains a reporting group that enables the labeled thiols to be detected [28, 29 and 30]. Then loss of this signal is assessed as an indication of protein thiol modification (top). Alternatively, unmodified protein thiols are blocked with an unlabeled alkylating reagent, often NEM, and then reversibly modified protein thiols are selectively reduced and labeled by reaction with a detectable thiol probe (middle) [31•• and 32••].