An important but understudied class of human exposures is comprised of reactive electrophiles that cannot be measured in vivo because they are short lived. An avenue for assessing these meaningful exposures focuses on adducts from reactions with nucleophilic loci of blood proteins, particularly Cys34 of human serum albumin, which is the dominant scavenger of reactive electrophiles in serum. We developed an untargeted analytical scheme and bioinformatics pipeline for detecting, quantitating and annotating Cys34 adducts in tryptic digests of human serum/plasma. The pipeline interrogates tandem mass spectra to find signatures of the Cys34-containing peptide, obtains accurate masses of putative adducts, quantitates adduct levels relative to a ‘housekeeping peptide’, and annotates modifications based on a combination of retention time, accurate mass, elemental composition and database searches. We used the adductomics pipeline to characterize 43 adduct features in archived plasma from healthy human subjects and found several that were highly associated with smoking status, race and other covariates. Since smoking is a strong risk factor for cancer and cardiovascular disease, our ability to discover adducts that distinguish smokers from nonsmokers with untargeted adductomics indicates that the pipeline is suitable for use in epidemiologic studies. In fact, adduct features were both positively and negatively associated with smoking, indicating that some adducts arise from reactions between Cys34 and constituents of cigarette smoke (e.g. ethylene oxide and acrylonitrile) while others (Cys34 oxidation products and disulfides) appear to reflect alterations in the serum redox state that resulted in reduced adduct levels in smokers.
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...
Small ubiquitin-like modifier (SUMO) modification of transcription factors is generally associated with repression. Reverse genetic analysis of SUMO-1, and -2 conserved residues emphasized the importance of dual charge reversals in abrogating the critical role of SUMO-2 K33, K35, and K42 in repression. GST-SUMO-2-affinity chromatography followed by liquid chromatography (LC)-MS analysis identified proteins that appeared to bind preferentially to WT SUMO-2 versus SUMO-2 K33E and K35E. LSD1, NXP-2, KIAA0809 (ARIP4), SAE2, RanGAP1, PELP1, and SETDB1 bound to SUMO-2 and not to SUMO-2 K33E, K42E, or K35E and K42E. Although LSD1 is a histone lysine demethylase, and histone H3K4 was demethylated at a SUMO-2-repressed promoter, neither overexpression of a dominant-negative LSD1 nor LSD1 depletion with RNA interference affected SUMO-2-mediated repression, indicating that LSD1 is not essential for repression, in this context. When tethered to a promoter by fusion to Gal4, NXP-2 repressed transcription, consistent with a role for NXP-2 in SUMO-mediated repression. SUMO-2-associated proteins identified in this study may contribute to SUMO-dependent regulation of transcription or other processes.regulation ͉ repressor ͉ ubiquitin-like S mall ubiquitin-like modifier (SUMO) is a ubiquitin-like protein that is reversibly covalently attached to lysines in target proteins, resulting in altered protein function, localization, or stability. SUMO modification of corepressors, coactivators, histones, histone-modifying enzymes, or sequence-specific transcription factors usually down-regulates transcription. For example, mutation so as to prevent SUMO modification increases transcription-factor activity associated with Elk-1, Sp3, c-myb, c-jun, AP2, or the p300 N terminus (1-5). In contrast to SUMO, ubiquitin modification is more frequently associated with proteasome-dependent degradation or activation of transcription. In fact, activation of transcription by the VP16 activation domain, in yeast, requires an E3 ubiquitin ligase (6). Addition of a SUMO consensus acceptor site to the Gal4-VP16 activation domain reduces transcription 10-fold (7). Thus, ubiquitin and SUMO modifications can have opposite effects on transcription.Despite these differences, SUMO-1, -2, and -3 are ubiquitinlike proteins and have ubiquitin-like structures. SUMO-2 and -3 are 95% identical and are 46% and 48%, respectively, identical to SUMO-1. In contrast, SUMO-1, -2, and -3 share only 18% identity with ubiquitin (Fig. 1). The differences in primary sequence and their role in the opposing transcriptional properties of SUMO and ubiquitin are being elucidated. SUMO interaction with histone deacetylases has been proposed as one mechanism of repression (1, 8). However, an unbiased directed screen to identify candidate SUMO-associated repressors has not been undertaken.The objective of our experiments was to identify proteins whose association with SUMO-2 depends on SUMO-2 residues required for repression. Such proteins would be candidate SUMO corepressors. We have u...
Xuanwei and Fuyuan counties in China have the highest lung cancer rates in the world due to household air pollution from combustion of smoky coal for cooking and heating. To discover potential biomarkers of indoor combustion products, we profiled adducts at the Cys34 locus of human serum albumin (HSA) in 29 nonsmoking Xuanwei and Fuyuan females who used smoky coal, smokeless coal or wood, and 10 local controls who used electricity or gas fuel. Our untargeted ‘adductomics’ method detected 50 tryptic peptides of HSA, containing Cys34 and prominent post-translational modifications. Putative adducts included Cys34 oxidation products, mixed disulfides, rearrangements and truncations. The most significant differences in adduct levels across fuel types were observed for S-glutathione (S-GSH) and S-γ-glutamylcysteine (S-γ-GluCys), both of which were present at lower levels in subjects exposed to combustion products than in controls. After adjustment for age and personal measurements of airborne benzo(a)pyrene, the largest reductions in levels of S-GSH and S-γ-GluCys relative to controls were observed for users of smoky coal, compared to users of smokeless coal and wood. These results point to possible depletion of GSH, an essential antioxidant, and its precursor γ-GluCys in nonsmoking females exposed to indoor-combustion products in Xuanwei and Fuyuan, China.
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