As DHFR contains five histidine residues that are distributed close to the active site and these loops, they can be used as probes to provide insights into the conformational changes ICI 182780 associated with ligand binding. The present results demonstrate the utility of His-HDX-MS for probing the microenvironment of histidine residues of proteins. This method can be used for studying important biological processes such as signal transduction, receptor-drug interactions, enzyme catalysis, protein folding, and opens the door for histidine scanning for investigating protein structure-function relationships. In the endoplasmic reticulum, critical protein maturation steps including N-glycosylation and disulfide-bond formation take place. Upon folding, native proteins can exit the ER by the secretory pathway, whereas misfolded proteins and incompletely assembled protein complexes are generally retained by a protein quality control machinery. Terminally misfolded proteins are degraded by the ER-associated degradation pathway, which involves the retrotranslocation of protein substrates to the cytosol and proteasomal degradation. To alleviate unfolding, ERAD substrates that contain disulfide bonds might also have to be reduced before retrotranslocation. Recently, ERdj5, a member of the protein disulfide isomerase family, has been demonstrated to facilitate this reduction step for certain ERAD substrates. The electron donor for ERdj5 remains to be identified, and neither is it clear whether ERdj5 is the only reducing PDI-family member involved in ERAD. In the endoplasmic reticulum, critical protein maturation steps including N-glycosylation and disulfide-bond formation take place. Upon folding, native proteins can exit the ER by the secretory pathway, whereas misfolded proteins and incompletely assembled protein complexes are generally retained by a protein quality control machinery. Terminally misfolded proteins are degraded by the ER-associated degradation pathway, which involves the retrotranslocation of protein substrates to the cytosol and proteasomal degradation. To alleviate unfolding, ERAD substrates that contain disulfide bonds might also have to be reduced before retrotranslocation. Recently, ERdj5, a member of the protein disulfide isomerase family, has been demonstrated to facilitate this reduction step for certain ERAD substrates. The electron donor for ERdj5 remains to be identified, and neither is it clear whether ERdj5 is the only reducing PDI-family member involved in ERAD. The experiment revealed one clear candidate interacting protein that was not recovered from control cell lysates. This protein had an apparent size of,90 kDa and contained endoglycosidase H -sensitive glycans indicating localization in the early secretory pathway. The interaction was not dependent on the formation of intermolecular disulfide bonds since the protein could be precipitated under reducing conditions. A similar result was obtained after pretreatment with the oxidant diamide.