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Gleason grading is an important prognostic indicator for prostate adenocarcinoma and is crucial for patient treatment decisions. However, intermediate-risk patients diagnosed in Gleason Grade Groups (GG) 2 and GG3 can harbour either aggressive or non-aggressive disease, resulting in under- or over-treatment of a significant number of patients. Here, we performed proteomic, differential expression, machine learning, and survival analyses for 1,348 matched tumour and benign sample runs from 278 patients. Three proteins (F5, TMEM126B and EARS2) were identified as candidate biomarkers in patients with biochemical recurrence. Multivariate Cox regression yielded 18 proteins, from which a risk score was constructed to dichotomise prostate cancer patients into low- and high-risk groups. This 18-protein signature is prognostic for the risk of biochemical recurrence and completely independent of the intermediate GG. Our results suggest that markers generated by computational proteomic profiling have the potential for clinical applications including integration into prostate cancer management.

SummaryThe proteome provides unique insights into biology and disease beyond the genome and transcriptome. Lack of large proteomic datasets has restricted identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types were analyzed by mass spectrometry. Deploying a clinically-relevant workflow to quantify 8,498 proteins, these data capture evidence of cell type and post-transcriptional modifications. Integrating multi-omics, drug response and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline revealed thousands of protein-specific biomarkers of cancer vulnerabilities. Proteomic data had greater power to predict drug response than the equivalent portion of the transcriptome. Further, random downsampling to only 1,500 proteins had limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger), available at https://cellmodelpassports.sanger.ac.uk, is a comprehensive resource revealing principles of protein regulation with important implications for future clinical studies.

The proteome provides unique insights into disease biology beyond the genome and transcriptome. A lack of large proteomic datasets has restricted the identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types are analyzed by mass spectrometry. Deploying a workflow to quantify 8,498 proteins, these data capture evidence of cell-type and post-transcriptional modifications. Integrating multi-omics, drug response, and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline reveals thousands of protein biomarkers of cancer vulnerabilities that are not significant at the transcript level. The power of the proteome to predict drug response is very similar to that of the transcriptome. Further, random downsampling to only 1,500 proteins has limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger) is a comprehensive resource available at https://cellmodelpassports.sanger.ac.uk.

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.

Reproducible research is the bedrock of experimental science. To enable the deployment of large-scale proteomics, we assess the reproducibility of mass spectrometry (MS) over time and across instruments and develop computational methods for improving quantitative accuracy. We perform 1560 data independent acquisition (DIA)-MS runs of eight samples containing known proportions of ovarian and prostate cancer tissue and yeast, or control HEK293T cells. Replicates are run on six mass spectrometers operating continuously with varying maintenance schedules over four months, interspersed with ~5000 other runs. We utilise negative controls and replicates to remove unwanted variation and enhance biological signal, outperforming existing methods. We also design a method for reducing missing values. Integrating these computational modules into a pipeline (ProNorM), we mitigate variation among instruments over time and accurately predict tissue proportions. We demonstrate how to improve the quantitative analysis of large-scale DIA-MS data, providing a pathway toward clinical proteomics., Nature Communications, 11 (1), ISSN:2041-1723

Motivation The output of electrospray ionization–liquid chromatography mass spectrometry (ESI-LC-MS) is influenced by multiple sources of noise and major contributors can be broadly categorized as baseline, random and chemical noise. Noise has a negative impact on the identification and quantification of peptides, which influences the reliability and reproducibility of MS-based proteomics data. Most attempts at denoising have been made on either spectra or chromatograms independently, thus, important 2D information is lost because the mass-to-charge ratio and retention time dimensions are not considered jointly. Results This article presents a novel technique for denoising raw ESI-LC-MS data via 2D undecimated wavelet transform, which is applied to proteomics data acquired by data-independent acquisition MS (DIA-MS). We demonstrate that denoising DIA-MS data results in the improvement of peptide identification and quantification in complex biological samples. Availability and implementation The software is available on Github (https://github.com/CMRI-ProCan/CRANE). The datasets were obtained from ProteomeXchange (Identifiers—PXD002952 and PXD008651). Preliminary data and intermediate files are available via ProteomeXchange (Identifiers—PXD020529 and PXD025103). Supplementary information Supplementary data are available at Bioinformatics online.

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 effects of compound loading on the identification of protein kinases (PKs) was examined using two previously reported sepharose-supported PK inhibitors (PKIs): bisindolylmaleimide X (S1) and CZC8004 (S2). Compound loadings of 0.1, 0.5, 2.5, 5, 10, 25, and 50% content and an ethanolamine-blocked control bead (no compound) were investigated. A 50% bead loading gave the highest level of PK identification for both S1 and S2, extracting 34 and 55 PKs, respectively, from a single cell lysate. Control beads allowed overall identification of 23 PKs, which we term the kinase beadome, whereas sepharose-supported sunitinib (S7; 50% loading) identified 20, 11 of which were common to the control beads. The reliability of bead pull-downs was examined in duplicate experiments using two independently synthesized batches each of S1 and S2. Bead S1 showed high similarity in the absolute numbers of PKs identified across two experiments, at 40 and 35 PKs, of which 26 were common across the two batches of beads, with 14 and 9 unique PKs identified in each experiment. The S2 beads extracted 61 and 64 PKs with 55 PKs common across the two bead batches examined. We also report on the development and use of a novel promiscuous PKI analogue, 2-[(5-chloro-2{[4-(piperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]-N-methylbenzene-sulfonamide (S15), which extracted 12 additional unique PKs over the two parent compounds from which it was designed, the combination of which identifies 160 unique PKs. S15 was based on the common pyrimidine core scaffold of S9 and S10. Thus, S15 expands the utility of kinobeads by broadening the kinome coverage for bead-based pull-down. Combining the data for all beads across 90 and 180 min liquid chromatography–mass spectrometry (LC–MS)/MS analysis identified a total of 160 unique PKs.

The cancer tissue proteome has enormous potential as a source of novel predictive biomarkers in oncology. Progress in the development of mass spectrometry (MS)-based tissue proteomics now presents an opportunity to exploit this by applying the strategies of comprehensive molecular profiling and big-data analytics that are refined in other fields of 'omics research. ProCan (ProCan is a registered trademark) is a program aiming to generate high-quality tissue proteomic data across a broad spectrum of cancer types. It is based on data-independent acquisition-MS proteomic analysis of annotated tissue samples sourced through collaboration with expert clinical and cancer research groups. The practical requirements of a high-throughput translational research program have shaped the approach that ProCan is taking to address challenges in study design, sample preparation, raw data acquisition, and data analysis. The ultimate goal is to establish a large proteomics knowledge-base that, in combination with other cancer 'omics data, will accelerate cancer research.

In the current study, we show how ProCan90, a curated data set of HEK293 technical replicates, can be used to optimize the configuration options for algorithms in the OpenSWATH pipeline. Furthermore, we use this case study as a proof of concept for horizontal scaling of such a pipeline to allow 45 810 computational analysis runs of OpenSWATH to be completed within four and a half days on a budget of US $10 000. Through the use of Amazon Web Services (AWS), we have successfully processed each of the ProCan 90 files with 506 combinations of input parameters. In total, the project consumed more than 340 000 core hours of compute and generated in excess of 26 TB of data. Using the resulting data and a set of quantitative metrics, we show an analysis pathway that allows the calculation of two optimal parameter sets, one for a compute rich environment (where run time is not a constraint), and another for a compute poor environment (where run time is optimized). For the same input files and the compute rich parameter set, we show a 29.8% improvement in the number of quality protein (2 peptide) identifications found compared to the current OpenSWATH defaults, with negligible adverse effects on quantification reproducibility or drop in identification confidence, and a median run time of 75 min (103% increase). For the compute poor parameter set, we find a 55% improvement in the run time from the default parameter set, at the expense of a 3.4% decrease in the number of quality protein identifications, and an intensity CV decrease from 14.0% to 13.7%.

We have developed a streamlined proteomic sample preparation protocol termed Accelerated Barocycler Lysis and Extraction (ABLE) that substantially reduces the time and cost of tissue sample processing. ABLE is based on pressure cycling technology (PCT) for rapid tissue solubilization and reliable, controlled proteolytic digestion. Here, a previously reported PCT based protocol was optimized using 1-4 mg biopsy punches from rat kidney. The tissue denaturant urea was substituted with a combination of sodium deoxycholate (SDC) and N-propanol. ABLE produced comparable numbers of protein identifications in half the sample preparation time, being ready for MS injection in 3 h compared with 6 h for the conventional urea based method. To validate ABLE, it was applied to a diverse range of rat tissues (kidney, lung, muscle, brain, testis), human HEK 293 cell lines, and human ovarian cancer samples, followed by SWATH-mass spectrometry (SWATH-MS). There were similar numbers of quantified proteins between ABLE-SWATH and the conventional method, with greater than 70% overlap for all sample types, except muscle (58%). The ABLE protocol offers a standardized, high-throughput, efficient, and reproducible proteomic preparation method that when coupled with SWATH-MS has the potential to accelerate proteomics analysis to achieve a clinically relevant turn-around time.

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 serine protease high-temperature requirement A (HtrA) (DegP) of the human pathogen Streptococcus pyogenes (group A Streptococcus; GAS) is localized to the ExPortal secretory microdomain and is reportedly essential for the maturation of cysteine protease streptococcal pyrogenic exotoxin B (SpeB). Here, we utilize HSC5 (M5 serotype) and the in-frame isogenic mutant HSC5DeltahtrA to determine whether HtrA contributes to the maturation of other GAS virulence determinants. Mutanolysin cell wall extracts and secreted proteins were arrayed by 2-DE and identified by MALDI-TOF PMF analysis. HSC5DeltahtrA had elevated levels of cell wall-associated M protein, whilst the supernatant had higher concentrations of M protein fragments and a reduced amount of mature SpeB protease, compared to wild-type (WT). Western blot analysis and protease assays revealed a delay in the maturation of SpeB in the HSC5DeltahtrA supernatant. HtrA was unable to directly process SpeB zymogen (proSpeB) to the active form in vitro. We therefore conclude that HtrA plays an indirect role in the maturation of cysteine protease SpeB.

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 final stage of mitosis is cytokinesis, which results in 2 independent daughter cells. Cytokinesis has 2 phases: membrane ingression followed by membrane abscission. IQGAP1 is a scaffold protein that interacts with proteins implicated in mitosis, including F-actin, myosin and CaM. IQGAP1 in yeast recruits actin and myosin II filaments to the contractile ring for membrane ingression. In contrast, we show that mammalian IQGAP1 is not required for ingression, but coordinates nuclear pore complex (NPC) reassembly and completion of abscission. Depletion of IQGAP1 disrupts Nup98 and mAb414 nuclear envelope localization and delays abscission timing. IQGAP1 phosphorylation increases 15-fold upon mitotic entry at S86, S330 and T1434, with the latter site being targeted by CDK2/Cyclin A and CDK1/Cyclin A/B in vitro. Expressing the phospho-deficient mutant IQGAP1-S330A impairs NPC reassembly in cells undergoing abscission. Thus, mammalian IQGAP1 functions later in mitosis than its yeast counterpart to regulate nuclear pore assembly in a S330 phosphorylation-dependent manner during the abscission phase of cytokinesis.

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.

The present study describes the isolation and pharmacological characterisation of the neurotoxin δ-missulenatoxin-Mb1a (δ-MSTX-Mb1a) from the venom of the male Australian eastern mouse spider, Missulena bradleyi. This toxin was isolated using reverse-phase high-performance liquid chromatography and was subsequently shown to cause an increase in resting tension, muscle fasciculation and a decrease in indirect twitch tension in a chick biventer cervicis nerve-muscle bioassay. Interestingly, these effects were neutralised by antivenom raised against the venom of the Sydney funnel-web spider Atrax robustus. Subsequent whole-cell patch-clamp electrophysiology on rat dorsal root ganglion neurones revealed that δ-MSTX-Mb1a caused a reduction in peak tetrodotoxin (TTX)-sensitive sodium current, a slowing of sodium current inactivation and a hyperpolarising shift in the voltage at half-maximal activation. In addition, δ-MSTX-Mb1a failed to affect TTX-resistant sodium currents. Subsequent Edman degradation revealed a 42-residue peptide with unusual N- and C-terminal cysteines and a cysteine triplet (Cys14-16). This toxin was highly homologous to a family of δ-atracotoxins (δ-ACTX) from Australian funnel-web spiders including conservation of all eight cysteine residues. In addition to actions on sodium channel gating and kinetics to δ-ACTX, δ-MSTX-Mb1a caused significant insect toxicity at doses up to 2000 pmol/g. δ-MSTX-Mb1a therefore provides evidence of a highly conserved spider δ-toxin from a phylogenetically distinct spider family that has not undergone significant modification.

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6–11 or pH 7–10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6–11 and novel pH 9–12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI ! 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) – tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI ! 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC ‘dimensions’ may be a useful complement to 2-DE for ‘near-to-total’ proteome coverage in the alkaline pH range.

While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M . pneumoniae and M . genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M . agalactiae and M . fermentans . The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M . fermentans and lipoprotein P48 of M . agalactiae . The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M . gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47 − mutant and wild-type M . gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47 , although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.

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 phospholipase A 2 inhibitor has been identified in the serum of the common tiger snake ( Notechis scutatus ). The inhibitor is composed of two chains, an α-chain and a β-chain, that form a non-covalently associated complex capable of inhibiting the enzymatic activity of all phospholipase A 2 enzymes it was tested against. The α and β-chains have been purified to homogeneity, digested and sequenced. From the peptide sequence generated, degenerate PCR primers were designed and used to elucidate the complete cDNA sequence of the chains using 5′ and 3′ RACE PCR. A total of three α-chain isoforms were identified, only one isoform of the β-chain was detected. The two-dimensional structure of the three α-chains and one β-chain were predicted using five prediction programs (discrimination of secondary structure class; nearest neighbour secondary structure, profile network from Heidelberg; self-optimised prediction method from multiple alignment, SSPAL). For each protein chain a consensus prediction was generated. Results are discussed in relation to the function of the protein, and how they may influence the three-dimensional structure of the inhibitor. Additionally, the sequences of several snake phospholipase A 2 inhibitors were used as the input for a motif prediction algorithm (MEME). The results are discussed in relation to the activity of these proteins.

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.

Although the resistance of snakes to their own venom is well known, until now no investigators have examined the serum of Australian snakes. Here we describe the identification and purification of a range of phospholipase A(2) (PLA(2)) inhibitors from the serum of Australian elapids. All PLA(2) inhibitors were composed of two protein chains, an alpha-chain and a beta-chain. The alpha-chains were approx. 22.5 kDa in size and variably glycosylated, whereas the beta-chains were approx. 19.8 kDa in size and not glycosylated. Identification of isoforms of the two subunit chains was significant because three of the six sera examined were from single snake specimens. In addition, the glycosylation patterns of the alpha-chains were thoroughly investigated in these unpooled sera. The functional and structural properties of the purified inhibitors were studied. Uniquely, a snake PLA(2) inhibitor was found to inhibit human type II PLA(2) enzyme, which has implications for the treatment of the many diseases in which PLA(2) enzymes have been implicated. Further, we demonstrate that the inhibitor forms a non-covalent association with a purified PLA(2) enzyme. Finally, the purified PLA(2) inhibitor was shown to protect in vivo against the lethal affects of a homologous PLA(2) enzyme, suggesting a role for PLA(2) inhibitors in the treatment of snake bite victims.

The phospholipase A2 enzyme, acanthoxin, found in the venom of the common death adder (Acanthophis antarcticus) as with other snake PLA2 enzymes displays neurotoxic activity. It is unclear whether this neurotoxic activity particu- lar to some snake PLA2 enzymes is a result of structural differences solely within the catalytic sites or at a distant location upon the molecules. We have predicted the three-dimensional structure of one of the two predominant isoforms of acanthoxin (A1) using comparative protein modeling tech- niques. Given the high degree of homology and the availability of a high quality crystallographic struc- ture, notexin was used as a molecular template to construct an all atom model of acanthoxin. The model was made using the program MODELLER3 and then refined with X-PLOR. Comparison be- tween the predicted structure of acanthoxin and several X-ray structures of toxic and nontoxic PLA2 enzymes has led to a testable two-step proposal of neurotoxic PLA2 activity; involving the favorable binding to acceptor molecules followed by enzy- matic intrusion upon the target membrane. The electrostatic potentials across the molecular sur- faces of toxic and nontoxic PLA2 enzymes were calculated (GRASP) and it was found that the toxic PLA2 enzymes possessed a charge distribution on the noncatalytic surface not identified in the non- toxic PLA2 enzymes. Thus we have identified resi- dues potentially involved in the interaction of the PLA2 enzymes with their acceptor molecules. Fur- thermore, the proposed acceptor molecule recogni- tion site is distant from the catalytic site which upon binding of the PLA2 to the acceptor molecule may enhance the enzymatic ability of the toxic PLA2 enzymes on particular cell types. Proteins 1999

Envenomation by Australian copperheads results mainly in muscle paralysis largely attributed to the presence of postsynaptic α-neurotoxins. However, poorly reversible neurotoxic effects suggest that these venoms may contain snake presynaptic phospholipase A2 neurotoxins (SPANs) that irreversibly inhibit neurotransmitter release. Using size-exclusion liquid chromatography, the present study isolated the first multimeric SPAN complex from the venom of the Australian common copperhead, Austrelaps superbus. The multimeric SPAN P-elapitoxin-As1a (P-EPTX-As1a) along with two novel monomeric SPANs and a new postsynaptic α-neurotoxin were then pharmacologically characterized using the chick biventer cervicis nerve-muscle preparation. All SPANs inhibited nerve-evoked twitch contractions at the neuromuscular junction without inhibiting contractile responses to cholinergic agonists or KCl. These actions are consistent with a prejunctional action to inhibit neurotransmitter release, without direct myotoxicity. Furthermore, the multimeric P-EPTX-As1a caused tetanic ‘fade’ in muscle tension under high frequency nerve stimulation, and produced a triphasic alteration to neurotransmitter release. These actions have been previously noted with other multimeric SPAN complexes such as taipoxin. Moreover, the neurotoxic α-subunit of P-EPTX-As1a shows high homology to taipoxin α-chain. Several other coagulopathic and myotoxic high mass proteins including a class PIII snake venom metalloproteinase, C-type lectin, l-amino acid oxidase, acetylcholinesterase and phospholipase B were also identified that may contribute to the overall toxicity of A. superbus venom. In conclusion, clinicians should be aware that early antivenom intervention might be necessary to prevent the onset of irreversible presynaptic neurotoxicity caused by multimeric and monomeric SPANs and that A. superbus venom is potentially capable of producing coagulopathic and myotoxic effects.

In contrast to all classical long-chain α-neurotoxins possessing the critical fifth disulfide bond, α-elapitoxin-Aa2a (α-EPTX-Aa2a), a novel long-chain α-neurotoxin from the common death adder Acanthophis antarcticus, lacks affinity for neuronal α7-type nicotinic acetylcholine receptors (nAChRs). α-EPTX-Aa2a (8850Da; 0.1-1μM) caused a concentration-dependent inhibition of indirect twitches, and blocked contractures to cholinergic agonists in the isolated chick biventer cervicis nerve-muscle preparation, consistent with a postsynaptic curaremimetic mode of action. α-EPTX-Aa2a (1-10nM) produced a potent pseudo-irreversible antagonism of chick muscle nAChRs, with an estimated pA(2) value of 8.311±0.031, which was not reversed by monovalent death adder antivenom. This is only 2.5-fold less potent than the prototypical long-chain α-neurotoxin, α-bungarotoxin. In contrast, α-EPTX-Aa2a produced complete, but weak, inhibition of (125)I-α-bungarotoxin binding to rat hippocampal α7 nAChRs (pK(I)=3.670), despite high sequence homology and similar mass to a wide range of long-chain α-neurotoxins. The mostly likely cause for the loss of α7 binding affinity is a leucine substitution, in loop II of α-EPTX-Aa2a, for the highly conserved Arg(33) in long-chain α-neurotoxins. Arg(33) has been shown to be critical for both neuronal and muscle activity. Despite this substitution, α-EPTX-Aa2a retains high affinity for muscle (α1)(2)βγδ nAChRs. This is probably as a result of an Arg(29) residue, previously shown to be critical for muscle (α1)(2)βγδ nAChR affinity, and highly conserved across all short-chain, but not long-chain, α-neurotoxins. α-EPTX-Aa2a therefore represents a novel atypical long-chain α-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes.

The human lens is an ideal tissue for the study of protein aging because crystallins are abundant and are present for a lifetime.1 Numerous posttranslational modifications to these polypeptides have been documented; the most abundant is deamidation.2,3 Deamidation of Asn and Gln residues has been described for many proteins,4,5 and it has been postulated that the introduction of a negative charge at a site that was formerly neutral may represent a “molecular clock” that could regulate protein turnover.6 To examine this, research has been undertaken with the aim of understanding the factors responsible for promoting deamidation of side chain amides.7 An examination of hundreds of peptides in which the residues flanking Asn and Gln were varied revealed that Gly, His, Glu, Ser, and Tyr facilitated deamidation. The mechanism of deamidation can involve a cyclic succinimide intermediate that, in the case of Asn, also leads to the formation of L-aspartyl, L-isoaspartyl, D-aspartyl, and D-isoaspartyl residues.8 Deamidation of crystallins has been documented,9–12 and several sites of Asp racemization in older lens crystallins have also been elucidated.13 Primary sequence is not the only factor determining the loss of ammonia from the amide residues of proteins. Secondary, tertiary, and quaternary structures significantly affect the rate of deamidation, and other factors may play a part within a biological environment. The extent of deamidation at particular sites in proteins can be predicted using computational means once crystal structures are available.6 Given that the hydrolysis of amides is time dependent, it could be predicted that proteins that are synthesized before birth and do not turnover may have few Asn/Gln residues adjacent to amino acids known to promote deamidation. The overall prevalence of Asn/Gln in crystallins is not significantly different from the average of all proteins.6 Deamidation of crystallins in older persons is detectable and may influence the properties of the human lens, as documented by David et al.,2 who showed that insoluble aggregates, which form progressively over time from soluble crystallins, have a higher degree of deamidation. It is not surprising that substantial deamidation of Gln and Asn would lead to protein unfolding. With the use of recombinant approaches, it has been demonstrated that the replacement of just one Asn by Asp in BB1-crystallin leads to measurable changes in physical properties, especially in the propensity of the protein to form aggregates.14 Deamidation of glutamines in βA3,15 βB1-crystallin,16 and γD-crystallin17 diminishes the stability of the proteins. Maintenance of the structural integrity of crystallins is becoming increasingly recognized as vital for the proper functioning of the lens and, therefore, vision.18–20 A major lens protein, α-crystallin, is a chaperone and binds other crystallins as they denature, forming high–molecular weight aggregates.21 Because there is no protein turnover in the center of the lens, after age 40 all the α-crystallin that was present at birth has been used in forming such aggregates. At this point, the stiffness of the lens increases dramatically,22 a phenomenon implicated in presbyopia—the inability to focus on near objects—that affects everyone once they reach age 45 to 50. Age-related nuclear cataract, a major cause of blindness, typically becomes noticeable a decade or more after presbyopia and is characterized by an even greater degree of protein modification such that in advanced cases, half the protein present in the lens center becomes colored, cross-linked, and insoluble in 8 M urea.23 It is unknown which factors are chiefly responsible for the progressive age-dependent denaturation of the crystallins in lenses. Heat is one possibility,24 and deamidation, if it were substantial, would be another that could lead to extensive unfolding. In this study we characterized the sites of deamidation in all the major crystallins in the human lens using tryptic digestion coupled with mass spectrometry. Analysis of the data revealed a novel sequence determinant implicated in protein deamidation. Because other tissues in the body25,26 that contain proteins are present for all our lives and organs with likely candidate polypeptides27–29 that remain to be investigated, this study also represents a step toward understanding the processes involved in age-dependent denaturation of these lifelong proteins and the consequences of this for human aging.

Loss of protein thiols is a key feature associated with the onset of age-related nuclear cataract (ARNC), however, little is known about the specific sites of oxidation of the crystallins. We investigated cysteine residues in ARNC lenses and compared them with age-matched normal lenses. Proteomic analysis of tryptic digests revealed ten cysteine residues in older normal lenses that showed no significant oxidation compared to foetal counterparts (Cys 170 in betaA1/3-crystallin, Cys 32 in betaA4-crystallin, Cys 79 in betaB1-crystallin, Cys 22, Cys 78/79, C153 in gammaC-crystallin and Cys 22, Cys 24 and Cys 26 in gammaS-crystallin). Although these thiols were not oxidised in normal lenses past the 6th decade, they were present largely as disulphides in the ARNC lenses. By contrast, two cysteine residues, Cys 41 in gammaC-crystallin and Cys 18 in gammaD-crystallin, were not oxidised, even in advanced ARNC lenses. These cysteines are buried deep within the protein and any unfolding associated with cataract must be insufficient to expose them to the oxidative environment present in the centre of advanced ARNC lenses. The vast majority of the loss of protein thiol observed in such lenses is due to disulphide bond formation.

The most complete proteome of human lenses has been compiled using 2-D LC-MS/MS analysis of foetal, aged normal and advanced nuclear cataract lenses. A total of 231 proteins were identified across all lens groups, including 112 proteins that have not been reported previously. Proteins were grouped according to their PANTHER molecular function classification in order to facilitate comparisons. Previously unreported N-terminal acetylation was detected in a number of proteins, with the majority being associated with the prior removal of a methionine residue. This pattern of proteolysis may indicate that methionine aminopeptidase activity is present in human lenses. Acetylation is likely to aid in the stability of proteins that are present in the lens for many decades. Protein sequences were also used to interrogate the three human lens cDNA libraries publicly available. Surprisingly, 84 proteins we identified were not present in the cDNA libraries.

The human eye is chronically exposed to light of wavelengths > 300 nm. In the young human lens, light of wavelength 300–400 nm is predominantly absorbed by the free Trp derivatives kynurenine (Kyn), 3-hydroxykynurenine (3OHKyn), and 3-hydroxykynurenine-O-β-D-glucoside (3OHKynG). These ultraviolet (UV) filter compounds are poor photosensitizers. With age, the levels of the free UV filters in the lens decreases and those of protein-bound UV filters increases. The photochemical behavior of these protein-bound UV filters and their role in UV damage are poorly elucidated and are examined here. UVA illumination of protein-bound UV filters generated peroxides (principally H2O2) in a metabolite-, photolysis-time-, and wavelength-dependent manner. Unmodified proteins, free Trp metabolites, and Trp metabolites that do not bind to lens proteins gave low peroxide yields. Protein-bound 3OHKyn (principally at Cys residues) yielded more peroxide than comparable Kyn and 3OHKynG adducts. Studies using D2O and sodium azide implicated 1O2 as a key intermediate. Illumination of the protein-bound adducts also yielded protein-bound Tyr oxidation products (DOPA, di-tyrosine) and protein cross-links via alternative mechanisms. These data indicate that the covalent modification of lens proteins by Kyn derivatives yields photosensitizers that may enhance oxidation in older lenses and contribute to age-related nuclear cataract.

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.

Major UV filters have been identified in the lens of the 13 lined ground squirrel (Spermophilus tridecemlineatus). These were found to be N-acetyl-3-hydroxykynurenine and N-acetyl-kynurenine, in addition to a small quantity of 3-hydroxykynurenine. The level of N-acetyl-3-hydroxykynurenine measured in the ground squirrel lens, 8.2mM, is approximately 11 times the concentration of 3-hyroxykynurenine glucoside reported previously for the human lens. Two additional UV filters of related structure were also present; however, their structures are still under investigation. HPLC elution profiles indicated that the ground squirrel lens cortex and nucleus contained comparable amounts of alpha-, beta(H)-, beta(L)-, and gamma-crystallins. Levels of GSH in the cortex and nucleus were 12.4 and 7.4mM, respectively. Such high concentrations of GSH may act to inhibit oxidation of the 3-hydroxykynurenine and N-acetyl-3-hydroxykynurenine. N-Acetylated kynurenines are less labile than those with free alpha-amino groups since N-acetyl-alpha-amino groups do not undergo spontaneous deamination. This modification thus stabilises the squirrel UV filters. In addition, because deamination is prevented, the decomposition products will not be involved in binding to lens proteins. Because of the similarity of the UV filters present in the ground squirrel to those in man, this species may be a suitable animal model for investigating the effects of UV radiation on cataract, and other ocular diseases, thought to involve exposure to light.

The present study describes the isolation and pharmacological characterisation of the neurotoxin delta-missulenatoxin-Mb1a (delta-MSTX-Mb1a) from the venom of the male Australian eastern mouse spider, Missulena bradleyi. This toxin was isolated using reverse-phase high-performance liquid chromatography and was subsequently shown to cause an increase in resting tension, muscle fasciculation and a decrease in indirect twitch tension in a chick biventer cervicis nerve-muscle bioassay. Interestingly, these effects were neutralised by antivenom raised against the venom of the Sydney funnel-web spider Atrax robustus. Subsequent whole-cell patch-clamp electrophysiology on rat dorsal root ganglion neurones revealed that delta-MSTX-Mb1a caused a reduction in peak tetrodotoxin (TTX)-sensitive sodium current, a slowing of sodium current inactivation and a hyperpolarising shift in the voltage at half-maximal activation. In addition, delta-MSTX-Mb1a failed to affect TTX-resistant sodium currents. Subsequent Edman degradation revealed a 42-residue peptide with unusual N- and C-terminal cysteines and a cysteine triplet (Cys(14-16)). This toxin was highly homologous to a family of delta-atracotoxins (delta-ACTX) from Australian funnel-web spiders including conservation of all eight cysteine residues. In addition to actions on sodium channel gating and kinetics to delta-ACTX, delta-MSTX-Mb1a caused significant insect toxicity at doses up to 2000 pmol/g. Delta-MSTX-Mb1a therefore provides evidence of a highly conserved spider delta-toxin from a phylogenetically distinct spider family that has not undergone significant modification.

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.