Quantitation of Nitration, Chlorination, and Oxidation in Hemoglobin of Breast Cancer Patients by Nanoflow Liquid Chromatography Tandem Mass Spectrometry

Abstract

Cells are continually exposed to endogenous reactive oxygen, nitrogen, and halogen species, causing damage to biomolecules. Among them, peroxynitrite and hypochlorous acid are not only oxidants but also biological nitrating and chlorinating agents, leading to the formation of 3-nitrotyrosine and 3-chlorotyrosine, respectively, in proteins. 3-Nitrotyrosine has been detected in vivo under several pathophysiological conditions, including breast cancer. Studies show that the concentrations of 3nitrotyrosine in plasma proteins and platelets were significantly elevated in breast cancer patients. Compared to blood serum albumin, hemoglobin adducts represent biomonitoring of exposure with a longer lifetime. In this study, human hemoglobin was freshly isolated from blood and digested into peptides with trypsin, and the levels of protein adducts, including nitration, nitrosylation, and chlorination of tyrosine as well as oxidation of methionine residues, were simultaneously quantified by nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) with selected reaction monitoring. The results demonstrated that the relative extents of nitration at α-Tyr-42 and β-Tyr-130, nitrosylation at α-Tyr-24, and chlorination at α-Tyr-24 and β-Tyr-130 are significantly higher in globin of 25 breast cancer patients compared to those in 25 healthy subjects (p < 0.05). In particular, nitration at α-Tyr-42 and chlorination at α-Tyr-24 showed the area under the receiver operating characteristic curve of >0.8. While the age of the subjects is correlated with the extents of some of these adducts, the body mass index does not have an effect on any of them. Starting with 1 drop of blood, our results indicated that this highly sensitive and specific nanoLC-NSI/MS/MS is useful in investigating the role of reactive nitrogen oxide species and reactive chlorine species in the etiology of breast cancer.

Characterization and Quantification of Acrolein-Induced Modifications in Hemoglobin by Mass Spectrometry─Effect of Cigarette Smoking

“…The rest of the chromatograms are shown in Figure S4 of the Supporting Information. When the methionine-containing peptides, such as α-Met-76 and β-Met-55, were chosen as the reference peptide, the corresponding methionine sulfoxide-containing peptides were included as the denominator [Figures C and S4­(A) of the Supporting Information] because the reported high levels of methionine sulfoxide in hemoglobin. ,,,, The integration of chromatographic peaks was performed by the perpendicular drop method. Table lists the SRM transitions of the 18 modified peptides, the six reference peptides, and two methionine-containing peptides.…”

“…Two microliters of each trypsin-digested globin sample from the subjects were injected into an UltiMate 3000 RSLCnano LC system (Dionex, Amsterdam, Netherlands) connected to an LTQ linear ion trap mass spectrometer (Thermo Electron Corp., San Jose, CA) and eluted under the conditions described previously. , Two time segments were arranged with 12 selected reaction monitoring (SRM) sets for elution between 19 and 40 min, and 15 SRM transitions were assigned to elution from 40 to 62 min, as listed in Table . Four synthetic peptides (31.0 pmol each) were analyzed in each preparation of the subject samples for calculation of digestion efficiency.…”

Mass Spectrometry Analysis of DNA and Protein Adducts as Biomarkers in Human Exposure to Cigarette Smoking: Acrolein as an Example

“…We have employed this NPR approach to various adducts (glutathionylation, nitration, nitrosylation, chlorination, oxidation, methylation, ethylation, carboxymethylation, and carboxyethylation, malondialdehyde, etc.) in human Hb to observe their relationship with smoking, oxidative stress, and diseases (diabetes and cancer). − The strategy is straightforward, without derivatization or enrichment by antibodies or affinity probes. The high sensitivity is attributed to the nanoflow LC and the fast scan rate of the ion trap mass spectrometer, albeit with the sacrifice of resolution .…”

Chloride Enhances DNA Reactivity with Chlorine under Conditions Relevant to Water Treatment

“…For example, extracellular bacterial DNA carrying antibiotic resistance genes is of interest in wastewater and surface water treatment . Furthermore, blood chloride concentrations normally range between 96 and 106 mM, and in vitro studies focus on the effects of endogenously produced HOCl on proteins and extracellular nucleic acids in blood matrices. , The extent to which Cl 2 contributes to degradation of these biomolecules is unclear and warrants further investigation.…”