Christopher H. Becker

Global changes to the ubiquitin system in Huntington's disease

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by expansion of CAG triplet repeats in the huntingtin (HTT) gene (also called HD) and characterized by accumulation of aggregated fragments of polyglutamine-expanded HTT protein in affected neurons. Abnormal enrichment of HD inclusion bodies with ubiquitin, a diagnostic characteristic of HD and many other neurodegenerative disorders including Alzheimer’s and Parkinson’s diseases, has suggested that dysfunction in ubiquitin metabolism may contribute to the pathogenesis of these diseases. Because modification of proteins with polyubiquitin chains regulates many essential cellular processes including protein degradation, cell cycle, transcription, DNA repair and membrane trafficking, disrupted ubiquitin signalling is likely to have broad consequences for neuronal function and survival. Although ubiquitin-dependent protein degradation is impaired in cell-culture models of HD and of other neurodegenerative diseases, it has not been possible to evaluate the function of the ubiquitin–proteasome system (UPS) in HD patients or in animal models of the disease, and a functional role for UPS impairment in neurodegenerative disease pathogenesis remains controversial. Here we exploit a mass-spectrometry-based method to quantify polyubiquitin chains and demonstrate that the abundance of these chains is a faithful endogenous biomarker of UPS function. Lys 48-linked polyubiquitin chains accumulate early in pathogenesis in brains from the R6/2 transgenic mouse model of HD, from a knock-in model of HD and from human HD patients, establishing that UPS dysfunction is a consistent feature of HD pathology. Lys 63- and Lys 11-linked polyubiquitin chains, which are not typically associated with proteasomal targeting, also accumulate in the R6/2 mouse brain. Thus, HD is linked to global changes in the ubiquitin system to a much greater extent than previously recognized.

Quantifying reproducibility for differential proteomics: noise analysis for protein liquid chromatography-mass spectrometry of human serum

SUMMARY Using replicated human serum samples, we applied an error model for proteomic differential expression profiling for a high-resolution liquid chromatography-mass spectrometry (LC-MS) platform. The detailed noise analysis presented here uses an experimental design that separates variance caused by sample preparation from variance due to analytical equipment. An analytic approach based on a two-component error model was applied, and in combination with an existing data driven technique that utilizes local sample averaging, we characterized and quantified the noise variance as a function of mean peak intensity. The results indicate that for processed LC-MS data a constant coefficient of variation is dominant for high intensities, whereas a model for low intensities explains Poisson-like variations. This result leads to a quadratic variance model which is used for the estimation of sample preparation noise present in LC-MS data.

Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools

The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.

Byonic: advanced peptide and protein identification software.

Byonic is the name of a software package for peptide and protein identification by tandem mass spectrometry. This software, which has only recently become commercially available, facilitates a much wider range of search possibilities than previous search software such as SEQUEST and Mascot. Byonic allows the user to define an essentially unlimited number of variable modification types. Byonic also allows the user to set a separate limit on the number of occurrences of each modification type, so that a search may consider only one or two chance modifications such as oxidations and deamidations per peptide, yet allow three or four biological modifications such as phosphorylations, which tend to cluster together. Hence, Byonic can search for tens or even hundreds of modification types simultaneously without a prohibitively large combinatorial explosion. Byonics Wildcard Search allows the user to search for unanticipated or even unknown modifications alongside known modifications. Finally, Byonics Glycopeptide Search allows the user to identify glycopeptides without prior knowledge of glycan masses or glycosylation sites. Curr. Protoc. Bioinform. 40:13.20.1‐13.20.14.

SINGLE NUCLEOTIDE POLYMORPHISM DETERMINATION USING PRIMER EXTENSION AND TIME-OF-FLIGHT MASS SPECTROMETRY

The high frequency of single nucleotide polymorphisms (SNPs) in the human genome makes them a valuable source of genetic markers for identity testing, genome mapping, and medical diagnostics. Conventional technologies for detecting SNPs are laborious and time‐consuming, often prohibiting large‐scale analysis. A rapid, accurate, and cost‐effective method is needed to meet the demands of a high‐throughput DNA assay. We demonstrate here that analysis of these genetic markers can now be performed routinely in a rapid, automated, and high‐throughput fashion using time‐of‐flight mass spectrometry and a primer extension assay with a novel cleavable primer. SNP genotyping by mass spectrometry involves detection of single‐base extension products of a primer immediately adjacent to the SNP site. Measurement of the mass difference between the SNP primer and the extension peak reveals which nucleotide is present at the polymorphic site. The primer is designed such that its extension products can be purified and chemically released from the primer in an automated format. The reduction in size of the products as a result of this chemical cleavage allows more accurate identification of the polymorphic base, especially in samples from a heterozygotic population. All six possible heterozygotes are resolved unambiguously, including an A/T heterozygote with extension products differing by only 9 Da. Multiplex SNP determination is demonstrated by simultaneously probing multiple SNP sites from a single polymerase chain reaction (PCR) product as well as from multiplexed PCR amplicons. Samples are processed in parallel on a robotic workstation, and analyzed serially in an automated mass spectrometer with analysis times of only a few seconds per sample, making it possible to process thousands of samples per day.

Protein Biomarker Identification in the CSF of Patients With CNS Lymphoma

PURPOSE Elucidation of the CSF proteome may yield insights into the pathogenesis of CNS disease. We tested the hypothesis that individual CSF proteins distinguish CNS lymphoma from benign focal brain lesions. METHODS We used a liquid chromatography/mass spectrometry-based method to differentially quantify and identify several hundred CSF proteins in CNS lymphoma and control patients. We used enzyme-linked immunosorbent assay (ELISA) to confirm one of these markers in an additional validation set of 101 cases. RESULTS Approximately 80 CSF proteins were identified and found to be present at significantly different concentrations, both higher and lower, in training and test studies, which were highly concordant. To further validate these observations, we defined in detail the expression of one of these candidate biomarkers, antithrombin III (ATIII). ATIII RNA transcripts were identified within CNS lymphomas, and ATIII protein was localized selectively to tumor neovasculature. Determination of ATIII concentration by ELISA was significantly more accurate (> 75% sensitivity; > 98% specificity) than cytology in the identification of cancer. Measurement of CSF ATIII levels was found to potentially enhance the ability to diagnose and predict outcome. CONCLUSION Our findings demonstrate for the first time that proteomic analysis of CSF yields individual biomarkers with greater sensitivity in the identification of cancer than does CSF cytology. We propose that the discovery of CSF protein biomarkers will facilitate early and noninvasive diagnosis in patients with lesions not amenable to brain biopsy, as well as provide improved surrogates of prognosis and treatment response in CNS lymphoma and brain metastasis.

Laser ablation of bulk YBa2Cu3O7−δ and cluster emission

Mass spectral investigations of ions produced directly from 1064‐nm plus 532‐nm laser beam ablation of a bulk high‐Tc superconducting YBa2Cu3O7−δ target reveal significant cluster emission as well as very intense low‐mass emission up to but below laser beam energy densities required to visually observe a plume of white light; at higher laser intensities, cluster emission essentially disappears while low‐mass ion intensities continue to grow. Cluster mass patterns ranging above 104 amu are observed and believed to be products of condensation rather than direct emission. Cluster ions higher in mass although not in distinguishable patterns, ranging beyond 106 amu, are observed, possibly due to direct emission. These results are relevant to high‐Tc superconducting thin‐film deposition by the laser ablation technique.

Organic compounds on crack surfaces in olivine from San Carlos, Arizona and Hualalai Volcano, Hawaii

Abstract Organic compounds associated with thin carbonaceous films on crack surfaces have been detected by thermal-desorption photoionization mass spectrometry in large single crystals of olivine from San Carlos, Arizona and Hualalai Volcano, Hawaii. Alkalis, silicon, aluminum and halogens are also present in the 3–4 nm thick carbonaceous films. The organics probably were not derived from the upper mantle or lower crust or from environmental biogenic contamination after eruption and cooling. It is likely that the carbonaceous films and organics were deposited or formed on crack surfaces created during eruption and cooling of the host alkali basalts. Whether the organics were produced abiotically by FischerTropsch-like reactions involving volcanic gases and fresh-fractured surfaces where reduced carbon was deposited, or whether the organics represent biogenic material that was assimilated into the magmatic system prior to or during magma ascent, cannot be ascertained at this time due to their low abundance.

Implication of perturbed axoglial apparatus in early pediatric multiple sclerosis.

Cerebrospinal fluid samples collected from children during initial presentation of central nervous system inflammation, who may or may not subsequently be diagnosed as having multiple sclerosis (MS), were subjected to large‐scale proteomics screening. Unexpectedly, major compact myelin membrane proteins typically implicated in MS were not detected. However, multiple molecules that localize to the node of Ranvier and the surrounding axoglial apparatus membrane were implicated, indicating perturbed axon–glial interactions in those children destined for diagnosis of MS. Ann Neurol 2012;

The fate of organic matter during planetary accretion: Preliminary studies of the organic chemistry of experimentally shocked murchison meteorite

It is possible that Earths biologic precursors were delivered by late-impacting asteroids or comets, and it is possible that these objects were a source of Earths volatile inventory. To understand the behavior of organic matter in carbonaceous meteorites during hypervelocity impact (1–2 km s−1), three samples of the Murchison (CM2) carbonaceous chondrite were shocked to 19, 20 and 36 GPa and analyzed by very sensitive thermal-desorption photoionization mass spectrometry (SALI). Thermal-desorption (25–800 °C) SALI mass spectra of unshocked Murchison reveal indigenous aliphatic, aromatic, sulfur and organosulfur compounds. Samples shocked to ≈20 GPa exhibit little or no loss of organic matter relative to the unshocked material. This is consistent with the earlier work of Tyburczyet al. (1986) which showed that incipient devolatilization of Murchison occurs at peak shock pressures near 20 GPa. The small amount of organic matter lost appears to have occurred by volatilization of elemental sulfur, amines and aliphatic compounds. In the sample shocked to 36 GPa, approximately 70% of the organic matter was volatilized as a result of impact. The residual organic matter desorbed at somewhat higher temperatures and displayed a different chemical signature. In particular, the shocked material has a lower alkene/alkane ratio than that of the starting material. The preliminary data suggest that it is unlikely that the indigenous organic matter in carbonaceous chondrite-like planetesimals could have survived impact on the Earth in the later stages of Earths accretion. However, chemical reactions that produce organic compounds with greater thermal stabilities may occur during impact or subsequent to impact by condensation of the impact-produced vapor plume.