Following digestion of all proteins with a peptidase such as trypsin, cysteine containing peptides are separated and identified by LC–MS and those containing modified thiols http://www.selleckchem.com/products/chir-99021-ct99021-hcl.html will appear as peak pairs corresponding to the isotopically light labeled thiol (unmodified) and the isotopically heavy labeled thiol (modified), separated by the mass difference between the probes. There are a number of advantages to this approach over gel-based methods. In addition
to identification of the thiol protein sensitive to a particular modification, the sensitive cysteine residue(s) can be determined. Furthermore, the use of two probes on an individual sample for analysis by LC–MS allows for internal comparison and can give a reliable measurement of the ratio of unmodified to modified cysteine. However, unlike gel-based methods where the background due to non-cysteine and unlabelled cysteine containing proteins is not an issue, the LC–MS analysis of complex samples would contain a significant background from these irrelevant sources. For this reason a labeling method using isotope coded affinity tag (ICAT) technology, which allows for affinity-purification of ICAT labeled peptides
before their separation and identification Cyclopamine cost by LC–MS is often a more robust approach [32••]. A recent extension of this approach is the development of cysteine tandem mass tags (cysTMTs) which allow for the selective isolation of modified cysteine peptides, as is the case in ICAT, as well as the potential for more clonidine accurate quantification by LC/MS/MS and the comparison of up to 6 conditions within a single experiment (http://www.piercenet.com). Although these methods greatly increase sensitivity by concentrating the selectively labeled peptide only, it is likely that affinity purification selects for the most abundant cysteine containing peptides and low abundance proteins could be left undetected. Many redox-active cysteine residues play central and varied roles in redox signaling pathways and in the control of redox homeostasis and the response to oxidative stress and xenobiotics. To identify
these cysteines and determine the functional significance of their modifications, a number of sensitive redox proteomic strategies have been developed. Using these approaches it is possible to identify those proteins that contain cysteine residues that are modified. In some cases it is also possible to determine the nature of the modification or at least indicate if the modification is reversible or irreversible, and perhaps identify the cysteine residue of interest. The use of LC/MS or LC/MS/MS can then enable the extent of the modification to be determined. However, as with all proteomic approaches, the methods outlined here only give a first indication that a particular condition affects thiols on a particular protein and perhaps a certain cysteine residue.