Mikel R. Roe

Targeted 18O-labeling for improved proteomic analysis of carbonylated peptides by mass spectrometry

Proteomic characterization of carbonylated amino acid sites currently relies on confidently matching tandem mass spectra (MS) to peptides within a sequence database. Although effective to some degree, reliable proteomic characterization of carbonylated peptides using this approach remains a challenge needing new, complementary solutions. To this end, we developed a method based on partial 18O-labeling of reactive carbonyl modifications, which produces a unique isotope signature in mass spectra of carbonylated peptides and enables their detection without reliance on matching MS2 spectra to a peptide sequence. Key to our method were optimized measures for eliminating trypsin-catalyzed incorporation of 18O at peptide C-termini, and for stabilizing the incorporated O within the carbonyl modification to prevent its loss during liquid chromatography separation. Applying our method to a rat skeletal muscle homogenate treated with the carbonyl modification 4-hyroxynonenal (4-HNE), we demonstrated its compatibility with solid-phase hydrazide enrichment of carbonylated peptides from complex mixtures. Additionally, we demonstrated the value of 18O isotope signatures for confirming HNE-modified peptide sequences matched via sequence database searching, and identifying modified peptides missed by MS2 and/or sequence database searching. Combining our 18O-labeling method with a customized automated software script, we systematically evaluated for the first time the efficiency of MS2 and sequence database searching for identifying HNE-modified peptides. We estimated that less than half of the modified peptides selected for MS2 were successfully identified. Collectively, our method and software should provide valuable new tools for investigators studying protein carbonylation via mass spectrometry-based proteomics.

Regioselective solvent-phase deuteration of polyphenolic compounds informs their identification by mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS) is a highly sensitive tool for the analysis of polyphenolic compounds in complex food and beverage matrices. However, the high degree of isomerism among polyphenols in general often complicates this approach, especially for identification of novel compounds. Here, we explore the utility of mild acid-catalyzed deuterium (MACD) labeling via electrophilic aromatic substitution as a complementary method for informing polyphenolic compound structure elucidation. To prevent hydrolysis of acid-labile glycosidic linkages, optimal reaction conditions that maximize regioselective hydrogen/deuterium (H/D) exchange of aromatic protons while preserving compound integrity were characterized (60°C, pH 3.0, 72 h). Under these conditions, standard compounds varying in the number and position of hydroxyl, glycosyl, and methyl groups about their aromatic core structure produced distinguishable H/D exchange patterns. The applicability of this method for the analysis of complex mixtures was demonstrated in red wine where the extent of deuterium exchange, together with accurate mass information, led to the putative identification of an unknown compound. The identification was further supported by tandem MS (MS/MS) data, which matched conclusively to the same compound in the Metlin LC-MS/MS library. With the capacity to discriminate between select isomeric forms, MACD labeling provides structural information that complements accurate mass and tandem mass spectral measurements for informing the identification of polyphenolics by MS.

Development of a Simple, Fast, and Accurate Method for the Direct Quantification of Selective Estrogen Receptor Modulators Using Stable Isotope Dilution Mass Spectrometry

A rapid analytical procedure was developed to quantify major selective estrogen receptor modulators (SERMs) simultaneously using stable isotope dilution mass spectrometry (SID-LCMS). Two novel isotopically labeled (SIL) analogues of natural SERMs, genistein and daidzein, were synthesized using a H/D exchange reaction mechanism. Computational chemistry coupled with MS and NMR data confirmed the site and mechanism of deuteration. The SIL analogues, which were mono- and dideutero substituted at the ortho positions, exhibited minimal deuterium isotope effects and were stable under the employed sample preparation protocol and MS analysis. An isotopic overlap correction was successfully employed to improve the accuracy and precision of the analytical method. The developed method, which was found to be sensitive, selective, precise and accurate, could be a valuable tool for research focused on determining the bioavailability of individual SERMs.

Targeted deuteration of polyphenolics for their qualitative and quantitative metabolomic analysis in plant-derived extracts

Polyphenolics are a chemically diverse class of plant specialized metabolites with strong antioxidant properties, and their consumption has been associated with improved human health. Metabolomic analysis of these compounds in both plant and mammalian samples has relied predominantly on liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS). Due to variable matrix effects across samples during ionization, the accuracy of this approach for quantifying compounds is greatly improved by incorporating stable isotope-labeled standards into the sample prior to analysis. However, commercially available, stable isotope-labeled, polyphenolic standards are both limited and costly. Here we present a protocol for generating stable isotope-labeled polyphenolics based on their deuteration by mild acid-catalyzed, electrophilic aromatic substitution. Importantly, this protocol is effective for generating stable isotope-labeled standards of many biologically relevant polyphenolics, both aglycones and the various conjugated forms alike.

Differential Accumulation and Degradation Of Anthocyanins In Red Norland Periderm is Dependent On Soil Type And Tuber Storage Duration

To determine how soil type, 2,4-dichlorophenoxyacetic acid (2,4-D) treatment, and storage affects color and anthocyanin accumulation of Red Norland potatoes, tubers were grown in sand or peat, with or without 2,4-D treatment, and measured at vine kill, harvest or after storage. Tubers grown in sand were less red and accumulated fewer anthocyanins than tubers grown in peat. 2,4-D treatment increased redness regardless of soil type. Redness loss varied greatly among tubers with storage. Tubers that lost color with storage had a two-fold reduction in anthocyanins, and a two-fold increase in benzoic and cinnamic acids compared to harvest, indicating chemical degradation of anthocyanidins via B-ring cleavage and autoxidation. Sand-grown potatoes did not exhibit greater cinnamic acids compared to peat-grown potatoes, suggesting that their color differences were due more to differences in biosynthesis than degradation during skin set. To improve Red Norland tuber color, research should focus on increasing biosynthesis of anthocyanins.ResumenPara determinar la influencia del tipo de suelo, el tratamiento con el ácido 2,4-diclorofenoxiacético (2,4-D) y el almacenamiento, sobre el color y acumulación de antocianinas en papa Red Norland, se sembraron tubérculos en arena o turba, con o sin tratamiento con 2,4-D, con mediciones al secado del follaje, a la cosecha, o después del almacenamiento. Los tubérculos que crecieron en la arena eran menos rojos y acumularon menos antocianinas que los cultivados en turba. El tratamiento con 2,4-D aumentó lo rojizo independientemente del tipo de suelo. La pérdida de la pigmentación roja varió grandemente entre los tubérculos con almacenamiento. Los tubérculos que perdieron el color en el almacén tuvieron el doble de reducción en antocianinas y un aumento al doble en ácidos benzoico y cinámico comparados a la cosecha, indicando degradación química de antocianidinas por vía del rompimiento del anillo B y por autooxidación. Las papas cultivadas en la arena no exhibieron mayores ácidos cinámicos en comparación con las cultivadas en la turba, lo que sugiere que sus diferencias en color se debieron más a diferencias en la biosíntesis que a la degradación durante el embarnecimiento de la piel. Para mejorar el color del tubérculo en Red Norland, la investigación debería enfocarse en el aumento de la biosíntesis de antocianinas.

216. Gene Therapy of Huntington Disease Using RNA Interference and Sleeping Beauty Transposon

Huntington Disease (HD) is a devastating neurologic disorder that is characterized by the abnormal expansion of a CAG repeat in the first exon of the huntingtin gene. This expansion results in the expression of a mutant huntingtin protein with an elongated polyglutamine stretch. The presence and aggregation of this mutant protein is responsible for the characteristic loss of the striatal neurons in HD leading to the clinically observed motor, psychiatric and cognitive deficits. Currently there is no effective treatment to prevent the neuronal loss associated with HD. The aim of this study was to evaluate an innovative gene therapy strategy combining RNA interference (RNAi) and gene transfer via the nonviral Sleeping Beauty (SB) transposon (Tn) system to mitigate the detrimental effects of the mutant huntingtin protein. While RNAi has been successfully used to suppress mammalian gene expression, a major shortcoming is the transient nature of the effect due to the intracellular degradation of the synthetic small interfering RNA (siRNA) molecules and/or the vector encoding the siRNA. By integration of the siRNA construct into the host genome via SB-mediated transposition, long-term suppression of gene expression can be achieved. SB is a functional vertebrate class II Tn element that mediates successful chromosomal insertion and long-term expression of its cargo transgenes in both cell culture and in vivo. We have constructed SB-Tns carrying siRNA transgenes targeting Exons 1, 7 and 62 of the human huntingtin gene. Real-time RT-PCR was used to quantify the huntingtin mRNA transcript levels in human neuroblastoma cells following transfection of the siRNA SB-Tns. In addition, DNA was isolated and subjected to bisulfite-mediated genomic sequencing to determine changes in methylation patterns. The results indicated that significant decreases up to 80% in huntingtin mRNA transcript levels relative to control were observed with several of the siRNA constructs. Interestingly, there was no evidence of increased methylation of CpGs in the regions of the hungtingtin sequence targeted by the siRNAs, suggesting that these specific siRNAs do not invoke epigenetic genomic modification. In addition, stereotaxic injection of an SB-Tn DsRed2 vector into the brains of HD transgenic mice resulted in significant expression of the reporter construct. In conclusion, huntingtin-specific siRNA constructs can down regulate huntingtin gene expression in cell culture. When delivered by the nonviral SB transposon, such constructs can potentially lead to long-term suppression of the mutant huntingtin gene expression, thus obliterating the harmful effects of the mutant protein. The combination of siRNAs, the SB transposon and an accurate transgenic mouse model now allows us to pursue a gene therapy approach for the treatment of HD.