Comprehensive Glycomic Profiling of Respiratory Tract Tissues of Tree Shrews by TiO2-PGC Chip Mass Spectrometry.

Due to its relatively small size, homology to humans, and susceptibility to human viruses, the tree shrew becomes an ideal alternative animal model for the study of human viral infectious diseases. However, there is still no report for the comprehensive glycan profile of the respiratory tract tissues in tree shrews. In this study, we characterized the structural diversity of N-glycans in the respiratory tract of tree shrews using our well-established TiO2-PGC chip-Q-TOF-MS method. As a result, a total of 219 N-glycans were identified. Moreover, each identified N-glycan was quantitated by a high sensitivity and accurate MRM method, in which 13C-labeled internal standards were used to correct the inherent run-to-run variation in MS detection. Our results showed that the N-glycan composition in the turbinate and lung was significantly different from the soft palate, trachea, and bronchus. Meanwhile, 28 high-level N-glycans in turbinate were speculated to be correlated with the infection of H1N1 virus A/California/04/2009. This study is the first to reveal the comprehensive glycomic profile of the respiratory tract of tree shrews. Our results also help to better understand the role of glycan receptors in human influenza infection and pathogenesis.

Age-Associated Methylation in Human Hemoglobin and Its Stability on Dried Blood Spots As Analyzed by Nanoflow Liquid Chromatography Tandem Mass Spectrometry.

Methylation of biomolecules is involved in many important biological processes. The contributing methylating agents arise from endogenous and exogenous sources (such as cigarette smoking). Human hemoglobin is easily accessible from blood and has been used as a molecular dosimeter for monitoring chemical exposure. We recently developed a method for characterization and quantification of the extents of methylation and ethylation in hemoglobin by nanoflow liquid chromatography tandem mass spectrometry under the selected reaction monitoring mode. Using this method, the relative extents of methylated and ethylated peptides in hemoglobin were quantified in nonsmoking subjects at various ages in this study. Among the nine methylation sites, we found that the extents of methylation were significantly higher in elderly subjects at the N-terminal and His-20 of α-globin, and at the N-terminal and Glu-26 of ß-globin. Moreover, the extents of methylation at these sites were significantly correlated with the age of the subjects. On the other hand, no statistically significant difference was found in the ethylated peptides. We also examined the stability of methylated and ethylated hemoglobin when stored on dried blood spot cards. The extents of these modifications on hemoglobin are stable for at least 4 weeks stored at room temperature. Our results suggest that age should be considered as a factor when measuring hemoglobin methylation and that dried blood spot is a valuable biomonitoring technique for hemoglobin modifications in epidemiological studies.

Simultaneous Mass Spectrometric Analysis of Methylated and Ethylated Peptides in Human Hemoglobin: Correlation with Cigarette Smoking.

Alkylating agents contained in cigarettes smoke might be related to cancer development. Post-translational protein methylation and ethylation may cause alteration of protein functions. Human hemoglobin (Hb) has been a target for molecular dosimetry because of its easy accessibility. The goal of this study is to investigate the relationship between the levels of methylation and ethylation at specific sites of Hb with smoking. Because of the low extent of modification of Hb isolated from blood, the methylation and ethylation sites were identified in Hb incubated with a methylating agent (methyl methanesulfonate, MMS) and ethylating agent (ethyl methanesulfonate, EMS), respectively, by accurate mass measurements. After trypsin digestion, the modification sites were identified by nanoflow LC-nanospray ionization coupled with high-resolution mass spectrometry. The selected reaction monitoring mode was used to quantify the relative extent of methylation and ethylation in human Hb incubated with MMS and EMS, respectively. Methylation occurred at 9 sites, including 1V, 20H, 50H, 72H of α-globin and 1V, 26E, 66K, 77H, 93C of ß-globin. Ethylation was detected at 11 sites, including 1V, 16K, 50H, 72H, 87H of α-globin and 1V, 17K, 66K, 77H, 92H, 93C of ß-globin. The relative extents of methylation and ethylation were measured in blood samples from 13 smokers and 13 nonsmokers. No statistically significant difference was found in the methylated peptides. On the other hand, the extents of ethylation at α-terminal Val, α-His-50, α-His-87, ß-terminal Val, ß-His-77, and ß-Cys-93 in Hb were significantly higher in smokers than in nonsmokers (p < 0.05). Furthermore, the relative extents of ethylation at these sites were statistically significantly correlated with the number of cigarettes smoked per day. Therefore, this assay, which requires as little as one drop of blood, should be helpful in measuring Hb ethylation as a potential biomarker for assessing the exposure to cigarette smoking.