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Mass spectrometry-based proteomics of individual ticks demonstrated persistence of mammalian host blood components, including α-and β-globin chains, histones, and mitochondrial enzymes, in Ixodes scapularis and Amblyomma americanum ticks for months after molting. Residual host proteins may identify sources of infection for ticks.W ithout transovarial or venereal transmission, a vector-borne pathogen's persistence in nature depends on successful passage between >1 species of vertebrate reservoirs. For Lyme borreliosis in eastern North America, black-legged tick (Ixodes scapularis) larvae acquire Borrelia burgdorferi from a reservoir host during their fi rst blood meal. Infection persists through subsequent molts, and when a tick feeds for the second time as a nymph it may transmit infection to another competent reservoir or to a human. Reservoir hosts for B. burgdorferi are commonly white-footed mice but also include chipmunks, voles, shrews, and ground-foraging birds.When a tick-borne agent has multiple reservoir hosts, assigning relative contributions of each species to maintenance of the pathogen in the environment may be diffi cult. One approach is to capture animals, sample blood or tissue for evidence of infection, and examine embedded ticks for the microorganism (1,2). However, this approach is laborand resource-intensive, and sample sizes are limited. Greater statistical power could be attained with fewer resources if questing ticks were examined not only for infection but also for the source of the last blood meal because the tick would likely have acquired the infection from that vertebrate. If the tick were engorged, this would be straightforward with the PCR, as demonstrated in mosquitoes (3,4). However, host-seeking nymphal hard ticks are fl at because their last blood meals were months earlier. Use of PCR to identify DNA of vertebrate mitochondria in ticks has been reported (5,6), but results lacked full sensitivity (7,8).An alternative approach is to detect residual proteins from the blood meal. Uptake and retention of host immunoglobulin into the hemolymph of different species of ticks have been documented (9), and Venneström and Jensen found vertebrate actin in I. ricinus nymphs weeks after the molt (10). Given these observations, we hypothesized that suffi cient host proteins remained in fl at ticks for identifi cation of blood meal by using proteins instead of DNA. The StudyWe used mass spectrometry (MS)-based proteomics, as described by Breci et al. and Koller et al. (11,12). Individual ticks or pools were pulverized after freezing in liquid nitrogen. Total proteins were precipitated in 95% ethanol at -20°C and recovered by centrifugation. Proteins of individual ticks were reduced with 100 mmol/L dithiothreitol, alkylated with 50 mmol/L iodoacetamide, digested with trypsin at a fi nal concentration of 0.01 μg/μL, and fi ltered through a C18 cartridge before being subjected to liquid chromatography (LC) with a 5%-50% acetonitrile gradient in 0.1% formic acid, followed by tandem MS (LC-MS/ MS; L...

Non-targeted analysis (NTA) encompasses a rapidly evolving set of mass spectrometry techniques aimed at characterizing the chemical composition of complex samples, identifying unknown compounds, and/or classifying samples, without prior knowledge regarding the chemical content of the samples. Recent advances in NTA are the result of improved and more accessible instrumentation for data generation and analysis tools for data evaluation and interpretation. As researchers continue to develop NTA approaches in various scientific fields, there is a growing need to identify, disseminate, and adopt community-wide method reporting guidelines. In 2018, NTA researchers formed the Benchmarking and Publications for Non-Targeted Analysis Working Group (BP4NTA) to address this need. Consisting of participants from around the world and representing fields ranging from environmental science and food chemistry to 'omics and toxicology, BP4NTA provides resources addressing a variety of challenges associated with NTA. Thus far, BP4NTA group members have aimed to establish a consensus on NTA-related terms and concepts and to create consistency in reporting practices by providing resources on a public Web site, including consensus definitions, reference content, and lists of available tools. Moving forward, BP4NTA will provide a setting for NTA researchers to continue discussing emerging challenges and contribute to additional harmonization efforts.

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We report a mass spectrometry-based comparative “bottom up” proteomics approach that combines d0/d4-succinic anhydride labeling with commercially available hydrazine (Hz)-functionalized beads (Affi-gel Hz beads) for detection, identification and relative quantification of site-specific oxylipid modifications in biological matrices. We evaluated and applied this robust and simple method for the quantitative analysis of oxylipid protein conjugates in cardiac mitochondrial proteome samples isolated from 3- and 24-month-old rat hearts. The use of d0/d4-succinic anhydride labeling, Hz-bead based affinity enrichment, nanoLC fractionation and MALDI-ToF/ToF tandem mass spectrometry yielded relative quantification of oxylipid conjugates with residue-specific modification information. Conjugation of acrolein (ACR), 4-hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-noneal (ONE) to cysteine, histidine and lysine residues were identified. HHE conjugates were the predominant subset of Michael-type adducts detected in this study. The HHE conjugates showed higher levels in mitochondrial preparations from young heart congruent with previous findings by others that the n-3/n-6 PUFA ratio is higher in young heart mitochondrial membranes. Although this study focuses on protein adducts of reactive oxylipids the method might be equally applicable to protein carbonyl modifications caused by metal catalyzed oxidation reactions.

BackgroundOsteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells.ResultsShotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines.ConclusionsThe results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

The isothiocyanate sulforaphane is a promising molecule for development as a therapeutic agent for patients with metastatic prostate cancer. Sulforaphane induces apoptosis in advanced prostate cancer cells, slows disease progression in vivo and is well tolerated at pharmacological doses. However, the underlying mechanism(s) responsible for cancer suppression remain to be fully elucidated. In this investigation we demonstrate that sulforaphane induces posttranslational modification of histone methyltransferase SUV39H1 in metastatic, androgen receptor-negative PC3 prostate cancer cells. Sulforaphane stimulates ubiquitination and acetylation of SUV39H1 within a C-terminal nuclear localization signal peptide motif and coincides with its dissociation from chromatin and a decrease in global trimethyl-histone H3 lysine 9 (H3K9me3) levels. Exogenous SUV39H1 expression leads to an increase in H3K9me3 and decreases sulforaphane-induced apoptotic signaling. SUV39H1 is thus identified as a novel mediator of sulforaphane cytotoxicity in PC3 cells. Our results also suggest SUV39H1 dynamics as a new therapeutic target in advanced prostate cancers.