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Atherosclerosis is a complex, multifactorial disease characterized by the buildup of plaque in the arterial wall. Apolipoprotein E gene deficient (Apoe-/-) mice serve as a commonly used tool to elucidate the pathophysiology of atherosclerosis because of their propensity to spontaneously develop arterial lesions. To date, however, an integrated omics assessment of atherosclerotic lesions in individual Apoe-/- mice has been challenging because of the small amount of diseased and nondiseased tissue available. To address this current limitation, we developed a multiomics method (Multi-ABLE) based on the proteomic method called accelerated Barocycler lysis and extraction (ABLE) to assess the depth of information that can be obtained from arterial tissue derived from a single mouse by splitting ABLE to allow for a combined proteomics-metabolomics-lipidomics analysis (Multi-ABLE). The new method includes tissue lysis via pressure cycling technology (PCT) in a Barocycler, followed by proteomic analysis of half the sample by nanoLC-MS and sequential extraction of lipids (organic extract) and metabolites (aqueous extract) combined with HILIC and reversed phase chromatography and time-of-flight mass spectrometry on the other half. Proteomic analysis identified 845 proteins, 93 of which were significantly altered in lesion-containing arteries. Lipidomic and metabolomic analyses detected 851 lipid and 362 metabolite features, which included 215 and 65 identified lipids and metabolites, respectively. The Multi-ABLE method is the first to apply a concurrent multiomics pipeline to cardiovascular disease using small (

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. It is generally diagnosed clinically after the irreversible loss of dopaminergic neurons and no general biomarkers currently exist. To gain insight into the underlying cellular causes of PD we aimed to quantify the proteomic differences between healthy control and PD patient cells.Sequential Window Acquisition of all THeoretical Mass Spectra was performed on primary cells from healthy controls and PD patients.In total, 1948 proteins were quantified and 228 proteins were significantly differentially expressed in PD patient cells. In PD patient cells, we identified seven significantly increased proteins involved in the unfolded protein response (UPR) and focused on cells with high and low amounts of PDIA6 and HYOU1. We discovered that PD patients with high amounts of PDIA6 and HYOU1 proteins were more sensitive to endoplasmic reticulum stress, in particular to tunicamycin. Data is available via ProteomeXchange with identifier PXD030723.This data from primary patient cells has uncovered a critical role of the UPR in patients with PD and may provide insight to the underlying cellular dysfunctions in these patients.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1–6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

The protein kinase inhibitor CTx-0152960 (6, 2-((5-chloro-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide), and the piperazinyl analogue, CTx-0294885 (7, 2-((5-chloro-2-((4-piperazin-1-ylphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide), were prepared using a hybrid flow and microwave approach. The use of flow chemistry approaches avoided the need for Boc-protection of piperidine in the key SNAr coupling with 1-fluoro-4-nitrobenzene. Microwave coupling of 4-morphilinoaniline 8 and 4-(piperazine-1-yl)aniline 9 with 2-(2,5-dichloropyrimidine-4-ylamino)-N-methylbenzamide 10, proved to be the most efficacious route to the target analogues 6 and 7. This hybrid methodology reduced the number of synthetic steps, gave enhanced overall yields and increased atom economy through step reduction and minimal requirement for chromatographic purification, relative to the original batch synthesis approach.

Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during sample preparation for proteomics. An alternative, 2-chloroacetamide, has recently been suggested to reduce the alkylation of residues other than cysteine, such as the N-terminus, Asp, Glu, Lys, Ser, Thr, and Tyr. Here we show that although 2-chloroacetamide reduces the level of off-target alkylation, it exhibits a range of adverse effects. The most significant of these is methionine oxidation, which increases to a maximum of 40% of all Met-containing peptides, compared with 2-5% with iodoacetamide. Increases were also observed for mono- and dioxidized tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The deleterious effects were observed for 2-chloroacetamide from three separate suppliers. The adverse impact of 2-chloroacetamide on methionine oxidation suggests that it is not the ideal alkylating reagent for proteomics.

The previous termvenomnext term of the European blackwidow previous termspidernext termLatrodectustredecimguttatus(Theridiidae) contains several high molecular mass (110–140 kDa) neurotoxins that induce neurotransmitter exocytosis. These includea vertebrate-specific α-previous termlatrotoxinnext term (α-LTX-Lt1a) responsible for the clinical symptoms of latrodectism andnumerous insect-specific latroinsectoxins (LITs). In contrast,little is known about the expression of these toxins in other Latrodectus species despite the fact that envenomation by these previous termspidersnext term induces previous termanext term similar clinical syndrome. Here we report highly conserved α-LTX, α-LIT and δ-LIT sequence tagsinL.mactans, L.hesperus and L. hasseltiprevious termvenomsnext term using tandem mass spectrometry,following bioassay-guidedseparation of previous termvenomsnext term by liquid chromatography. Despite this sequence similarity, we show that the anti-α-LTX monoclonal antibody 4C4.1, raised against α-LTX-Lt1a,fails to neutralize the neurotoxicity of all other Latrodectus venomstested in an isolated chick biventer cervicis nerve-muscle bioassay. This suggests that there are important structural differences between α-LTXs in theridiid previous termspidernext termprevious termvenomsnext term. We therefore cloned and sequenced the α-LTX from the Australian previous termrednext term-previous termbacknext termprevious termspidernext termL. hasselti (α-LTX-Lh1a). The deduced amino acid sequence of the mature α-LTX-Lh1acomprises 1180 residues(∼132 kDa) with ∼93%sequenceidentity with α-LTX-Lt1a. α-LTX-Lh1a is composed of an N-terminal domainand previous terma central region containing22 ankyrin-like repeats. The presence of two furin cleavage sites,conserved with α-LTX-Lt1a, indicates thatα-LTX-Lh1ais derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show thatα-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody.

Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO2 chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged–dynamin II–proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.

The urea-soluble proteins from the nucleus of two young, two aged, and two early-stage nuclear cataract lenses were subjected to tryptic digestion and analysis by 2D LC-MS/MS. Several novel post-translational modifications were identified. Deamidation was, by far, the most common modification. A number of differences were found in cataract compared to normal lenses, most notably an increase in the number of oxidized tryptophan residues. Semiquantitative analysis revealed that there appeared to be a trend toward increased levels of deamidation with age; however, there was no apparent increase upon the onset of nuclear cataract. This is in contrast to Trp oxidation, where an increase in the extent of modification was apparent in cataract lenses when compared to aged normal lenses. These findings suggest Trp oxidation may be involved in nuclear cataract development.

The omega-atracotoxins (omega-ACTX) are a family of arthropod-selective peptide neurotoxins from Australian funnel-web spider venoms (Hexathelidae: Atracinae) that are candidates for development as biopesticides. We isolated a 37-residue insect-selective neurotoxin, omega-ACTX-Ar1a, from the venom of the Sydney funnel-web spider Atrax robustus, with high homology to several previously characterized members of the omega-ACTX-1 family. The peptide induced potent excitatory symptoms, followed by flaccid paralysis leading to death, in acute toxicity tests in house crickets. Using isolated smooth and skeletal nerve-muscle preparations, the toxin was shown to lack overt vertebrate toxicity at concentrations up to 1 microM. To further characterize the target of the omega-ACTXs, voltage-clamp analysis using the whole-cell patch-clamp technique was undertaken using cockroach dorsal unpaired median neurons. It is shown here for the first time that omega-ACTX-Ar1a, and its homolog omega-ACTX-Hv1a from Hadronyche versuta, reversibly block both mid-low- (M-LVA) and high-voltage-activated (HVA) insect calcium channel (Ca(v)) currents. This block occurred in the absence of alterations in the voltage-dependence of Ca(v) channel activation, and was voltage-independent, suggesting that omega-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1 microM omega-ACTX-Ar1a failed to significantly affect global K(v) channel currents. However, 1 microM omega-ACTX-Ar1a caused a modest 18% block of insect Na(v) channel currents, similar to the minor block of Na(v) channels reported for other insect Ca(v) channel blockers such as omega-agatoxin IVA. These findings validate both M-LVA and HVA Ca(v) channels as potential targets for insecticides.

4-(2-Aminophenyl)-4-oxobutanoic acid, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid and glutathionyl-kynurenine have been identified as novel metabolites in normal and cataractous human lenses following total synthesis and comparison with authentic human lens samples. Their structures are consistent with those derived from the major human lens UV filters kynurenine and 3-hydroxykynurenine, and it is proposed that these compounds also play a role as UV filters. These metabolites were isolated in pmol/mg levels (dry mass) in lenses. 4-(2-Amino-3-hydroxyphenyl)-4-oxobutanoic acid and glutathionyl-kynurenine were found to be unstable at physiological pH. Other potential metabolites, glutathionyl-3-hydroxykynurenine, kynurenine yellow and 3-hydroxykynurenine yellow, were not detected in either normal or cataractous lenses.

The alpha-, beta-, and gamma-crystallins are the major structural proteins of mammalian lenses. The human lens also contains tryptophan-derived UV filters, which are known to spontaneously deaminate at physiological pH and covalently attach to lens proteins. 3-Hydroxykynurenine (3OHKyn) is the third most abundant of the kynurenine UV filters in the lens, and previous studies have shown this compound to be unstable and to be oxidized under physiological conditions, producing H2O2. In this study, we show that methionine and tryptophan amino acid residues are oxidized when bovine alpha-crystallin is incubated with 3-hydroxykynurenine. We observed almost complete oxidation of methionines 1 and 138 in alphaA-crystallin and a similar extent of oxidation of methionines 1 and 68 in alphaB-crystallin after 48 h. Tryptophans 9 and 60 in alphaB-crystallin were oxidized to a lesser extent. AlphaA-crystallin was also found to have 3OHKyn bound to its single cysteine residue. Examination of normal aged human lenses revealed no evidence of oxidation of alpha-crystallin; however, oxidation was detected at methionine 1 in both alphaA- and alphaB-crystallin from human cataractous lenses. Age-related nuclear cataract is associated with coloration and insolubilization of lens proteins and extensive oxidation of cysteine and methionine residues. Our findings demonstrate that 3-hydroxykynurenine can readily catalyze the oxidation of methionine residues in both alphaB- and alphaA-crystallin, and it has been reported that alpha-crystallin modified in this way is a poorer chaperone. Thus, 3-hydroxykynurenine promotes the oxidation and modification of crystallins and may contribute to oxidative stress in the human lens.

Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the beta-tubulin subunit of alpha/beta-tubulin heterodimers, and they act to inhibit cell division by disrupting microtubule dynamics. Despite the effectiveness of these agents, drug resistance is a major clinical problem. To identify the underlying mechanisms behind vinca alkaloid resistance, we have performed high resolution differential proteome analysis. Treatment of drug-sensitive human leukemia cells (CCRF-CEM) with vincristine identified numerous proteins involved in the cellular response to vincristine. In addition, differential protein expression was analyzed in leukemia cell lines selected for resistance to vincristine (CEM/VCR R) and vinblastine (CEM/VLB100). This combined proteomic approach identified 10 proteins altered in both vinca alkaloid response and resistance: beta-tubulin, alpha-tubulin, actin, heat shock protein 90beta, 14-3-3tau, 14-3-3epsilon, L-plastin, lamin B1, heterogeneous nuclear ribonuclear protein-F, and heterogeneous nuclear ribonuclear protein-K. Several of these proteins have not previously been associated with drug resistance and are thus novel targets for elucidation of resistance mechanisms. In addition, seven of these proteins are associated with the tubulin and/or actin cytoskeletons. This study provides novel insights into the interrelationship between the microtubule and microfilament systems in vinca alkaloid resistance.

UV light has often been investigated as a risk factor for the most common cause of blindness, human age-related cataract. One mechanism whereby UV light could induce cataract is via the action of photosensitisers. In this regard, xanthurenic acid has recently been highlighted since it has been reported to be present in the human lens and, in model studies, it markedly enhances the photo-oxidation of proteins by wavelengths of light that penetrate the cornea. In this study we used HPLC and mass spectrometry to examine whether xanthurenic acid is indeed present in human lenses and, if so, the effect of age on its lenticular concentration. Xanthurenic acid could be formed artefactually by incubation of 3-hydroxykynurenine (3OHKyn) yellow, a known autoxidation product of the lenticular UV filter, 3OHKyn, in the presence of air and light, however, it could not be detected in any human lenses studied. Therefore, it appears unlikely that xanthurenic acid plays a role in lens aging or human cataract.

Two complementary approaches were used to determine the effects of heat shock on wheat-grain quality. Heat-susceptible (cv. Wyuna) and heat-tolerant (cv. Fang) cultivars of wheat were compared after heat shock, utilising both dough-quality testing and proteome analysis. Plants were grown at day/night temperatures of 24/18 °C during development, with stressed plants being subjected to heat shock at day/night temperatures of 40/25 °C on 15, 16 and 17 days post-anthesis. Grain samples were taken during development (17 days post-anthesis) and at maturity (45 days post-anthesis). Dough-quality testing of flour indicated that the Wyuna cultivar exhibited a decrease in dough strength after heat shock, whilst the Fang cultivar exhibited an increase. Proteome studies conducted on endosperm (at 17 days post-anthesis) showed that the heat-tolerant Fang cultivar exhibited a stronger and more diverse «heat shock response» than Wyuna. In total, 48 protein spots exhibiting differential expression between control and heat shock treatments were excised from gels and analysed by mass spectrometry. The resultant tryptic-peptide mass fingerprint data was submitted to SWISS-PROT and TrEMBL databases for protein identification. The majority of heat-shock associated proteins had low molecular mass and showed database similarity to previously characterised small heat shock proteins. Several discrete isoforms of the low molecular weight heat shock proteins were observed as differentially expressed between the two cultivars. Furthermore, seven protein spots were expressed in heat shocked Fang but not in heat shocked Wyuna, thus were further characterised utilising tandem mass spectrometry. These possible marker proteins for heat-tolerance may assist breeders in the selection of heat-tolerant cultivars that would not be expected to lose dough strength in such an environment.

A total of 220 cell envelope-associated proteins were successfully extracted and separated from Trichoderma reesei mycelia actively synthesizing and secreting proteins and from mycelia in which the secretion of proteins are low. Altogether 56 spots were examined by nanoelectrospray tandem mass spectrometry and amino acid sequence was obtained for 32 spots. From these, 20 spots were identified by Advanced BLAST searches against all databases available to BLAST. The most abundant protein in both types of mycelia was HEX1, the major protein in Woronin body, a structure unique to filamentous fungi. Other proteins identified were vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta. Partial short amino acid sequence obtained from some proteins did not allow them to be assigned to a specific protein in the database by BLAST search. In some cases, the tandem mass spectrometry spectra were too complicated to be able to assign an amino acid sequence with certainty. The number of spots (12) giving a clear signal but finding no match in the databases suggests that a majority of proteins associated with a filamentous fungal cell wall, are novel. Some technical problems related to protein isolation are also discussed.

The identification and characterisation of Monodelphis proteins has required cross-species analysis. Protein expression was investigated in normal, nonirradiated adult fibroblasts and also in fibroblastic cells from a benign cutaneous tumour after chronic ultraviolet (UVB) exposure and a metastatic cutaneous tumour after intermittent exposure. Proteins were separated and visualised by two-dimensional gel electrophoresis (2-D PAGE) and a peptide mass fingerprint (PMF) was obtained for protein spots using matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDITOF-MS). Cross-species PMF database analysis facilitated the identification of 120 proteins, constituting 46.5% of the proteins analysed. The identification of two proteins was confirmed by internal amino acid sequencing using tandem MS. Differential protein expression was observed between normal fibroblasts and those in tumours chronically or intermittently exposed. A number of tropomyosin and vimentin isoforms were expressed only in cells from the metastatic tumour induced by intermittent exposure to UV radiation. These results highlight the value of cross-species PMF analysis for the rapid characterisation of proteins from a poorly defined species and also show how proteomics can be used to detect changes in protein expression in differentially treated cells.

Many low molecular weight substances in human tears, including protein and lipid species, have yet to be characterized. Some of these uncharacterized substances may well be important in the pathogenesis of ocular surface disease or in ocular discomfort. The aim of this study was to build a biochemical profile of low molecular weight species in tears, and to determine its repeatability. A total of 80 tear samples were collected from 11 subjects. Tear samples were dialysed to remove salts, added to a matrix of α-cyano-4- hydroxycinnamic acid, and analysed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Species were separated based on their mass to charge ratio (m : z). The repeatability of the appearance of the different species was analysed using logistic regression and diurnal and day-to-day repeatability were ascertained. Peptides were identified in the range of 848–3897 Da. Of these, 39 peptides were found to be present in more than 10 / 80 samples. There was no diurnal variation in the peptides. All species were found to occur repeatably, with the exception of peptide 1653 Da. This study has demonstrated that the majority of low molecular weight species in tears are repeatably present and do not exhibit diurnal variation. Further study aims to characterize these species and to identify changes in tear profiles between subject groups.