A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys 34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSACys 34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys 34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys 34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ؎2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected band y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.004606, 1-13, 2011.Reactive electrophiles, particularly aldehydes, epoxides, quinones, and short-lived oxygen and nitrogen species, have long been implicated as causes of major human diseases (1-5). These electrophiles enter the blood following metabolism of exogenous compounds, from oxidation of lipids and other natural molecules, from ionizing radiation, and from inflammation associated with infections and disease processes. Because of their short life spans in vivo, it is rarely possible to measure reactive electrophiles in body fluids. However, adducts produced by reactions with blood nucleophiles, notably hemoglobin (Hb) 1 and human serum albumin (HSA), reflect the presence of reactive electrophiles in the systemic circulation (6, 7). Levels of targeted Hb and/or HSA adducts have been investigated in human blood for several exogenous or endogenous toxicants that are either electrophilic carcinogens or their precursors, notably, acrylamide, aflatoxin B1, benzene, 1,3-butadiene, ethylene oxide, as well as assorted aromatic amines, polycyclic aromatic hydrocarbons, and aldehyde...
