Electron-Activated Dissociation and Collision-Induced Dissociation Glycopeptide Fragmentation for Improved Glycoproteomics.

Tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) has proven a versatile tool for the identification and quantification of proteins and their post-translational modifications (PTMs). Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterization of site-specific glycosylation of proteins remains analytically challenging. Collision-induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but the loss of labile modifications renders CID inappropriate for detailed characterization of site-specific glycosylation. Electron-based dissociation methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localization, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron-activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalian O- and N-glycopeptides. We found CID fragmentation identified the most glycopeptides and generally produced higher quality spectra, but EAD provided improved confidence in glycosylation site localization. Supplementing EAD with CID fragmentation (EAciD) further increased the number and quality of glycopeptide identifications, while retaining localization confidence. These methods will be useful for glycoproteomics workflows for either optimal glycopeptide identification or characterization.

Non-targeted proteomics reveals altered immune response in geographically distinct populations of green sea turtles (Chelonia mydas).

All seven species of sea turtle are facing increasing pressures from human activities that are impacting their health. Changes in circulating blood proteins of an individual, or all members of a population, can provide an early indicator of adverse health outcomes. Non-targeted measurement of all detectable proteins in a blood sample can indicate physiological changes. In the context of wildlife toxicology, this technique can provide a powerful tool for discovering biomarkers of chemical exposure and effect. This study presents a non-targeted examination of the protein abundance in sea turtle plasma obtained from three geographically distinct foraging populations of green turtles (Chelonia mydas) on the Queensland coast. Relative changes in protein expression between sites were compared, and potential markers of contaminant exposure were investigated. Blood plasma protein profiles were distinct between populations, with 85 out of the 116 identified proteins differentially expressed (p < 0.001). The most strongly dysregulated proteins were predominantly acute phase proteins, suggestive of differing immune status between the populations. The highest upregulation of known markers of immunotoxicity, such as pentraxin fusion and complement factor h, was observed in the Moreton Bay turtles. Forty-five different organohalogens were also measured in green turtle plasma samples as exposure to some organohalogens (e.g., polychlorinated biphenyls) has previously been identified as a cause for immune dysregulation in marine animals. The few detected organohalogens were at very low (pg/mL) concentrations in turtles from all sites, and are unlikely to be the cause of the proteome differences observed. However, the changes in protein expression may be indicative of exposure to other chemicals or environmental stressors. The results of this study provide important information about differences in protein expression between different populations of turtles, and guide future toxicological and health studies on east-Australian green sea turtles.

N-glycosylation on Oryza sativa root germin-like protein 1 is conserved but not required for stability or activity.

Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251 ± 70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.

Serum proteomes of Santa Gertrudis cattle before and after infestation with Rhipicephalus australis ticks.

Previous studies have applied genomics and transcriptomics to identify immune and genetic markers as key indicator traits for cattle tick susceptibility/resistance; however, results differed between breeds, and there is lack of information on the use of host proteomics. Serum samples from Santa Gertrudis cattle (naïve and phenotyped over 105 days as tick-resistant [TR] or tick-susceptible [TS]) were used to conduct differential abundance analyses of protein profiles. Serum proteins were digested into peptides followed by identification and quantification using sequential window acquisition of all instances of theoretical fragment ion mass spectrometry. Before tick infestation, abundance of 28 proteins differed significantly (adjusted P < 10-5 ) between TR and TS. These differences were also observed following tick infestation (TR vs TS) with a further eight differentially abundant proteins in TR cattle, suggesting possible roles in adaptive responses. The intragroup comparisons (TS-0 vs TS and TR-0 vs TR) showed that tick infestation elicited quite similar responses in both groups of cattle, but with relatively stronger responses in TR cattle. Many of the significantly differentially abundant proteins in TR Santa Gertrudis cattle (before and after tick infestation) were associated with immune responses including complement factors, chemotaxis for immune cells and acute-phase responses.

Structural insights into the mechanism of inhibition of AHAS by herbicides.

Acetohydroxyacid synthase (AHAS), the first enzyme in the branched amino acid biosynthesis pathway, is present only in plants and microorganisms, and it is the target of >50 commercial herbicides. Penoxsulam (PS), which is a highly effective broad-spectrum AHAS-inhibiting herbicide, is used extensively to control weed growth in rice crops. However, the molecular basis for its inhibition of AHAS is poorly understood. This is despite the availability of structural data for all other classes of AHAS-inhibiting herbicides. Here, crystallographic data for Saccharomyces cerevisiae AHAS (2.3 Å) and Arabidopsis thaliana AHAS (2.5 Å) in complex with PS reveal the extraordinary molecular mechanisms that underpin its inhibitory activity. The structures show that inhibition of AHAS by PS triggers expulsion of two molecules of oxygen bound in the active site, releasing them as substrates for an oxygenase side reaction of the enzyme. The structures also show that PS either stabilizes the thiamin diphosphate (ThDP)-peracetate adduct, a product of this oxygenase reaction, or traps within the active site an intact molecule of peracetate in the presence of a degraded form of ThDP: thiamine aminoethenethiol diphosphate. Kinetic analysis shows that PS inhibits AHAS by a combination of events involving FAD oxidation and chemical alteration of ThDP. With the emergence of increasing levels of resistance toward front-line herbicides and the need to optimize the use of arable land, these data suggest strategies for next generation herbicide design.

Commercial AHAS-inhibiting herbicides are promising drug leads for the treatment of human fungal pathogenic infections.

The increased prevalence of drug-resistant human pathogenic fungal diseases poses a major threat to global human health. Thus, new drugs are urgently required to combat these infections. Here, we demonstrate that acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway, is a promising new target for antifungal drug discovery. First, we show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of Candida albicans AHAS (Ki values as low as 800 pM) and have determined high-resolution crystal structures of this enzyme in complex with several of these herbicides. In addition, we have demonstrated that chlorimuron ethyl (CE), a member of the sulfonylurea herbicide family, has potent antifungal activity against five different Candida species and Cryptococcus neoformans (with minimum inhibitory concentration, 50% values as low as 7 nM). Furthermore, in these assays, we have shown CE and itraconazole (a P450 inhibitor) can act synergistically to further improve potency. Finally, we show in Candida albicans-infected mice that CE is highly effective in clearing pathogenic fungal burden in the lungs, liver, and spleen, thus reducing overall mortality rates. Therefore, in view of their low toxicity to human cells, AHAS inhibitors represent a new class of antifungal drug candidates.

Evolution for improved secretion and fitness may be the selective pressures leading to the emergence of two NDM alleles.

The New Delhi metallo-ß-lactamase (NDM-1) mediates resistance to ß-lactam antibiotics. NDM-1 was likely formed as the result of a gene fusion between sequences encoding the first six amino acids of cytoplasm-localised aminoglycosidase, AphA6, and a periplasmic metallo-ß -lactamase. We show that NDM-1 has an atypical signal peptide and is inefficiently secreted. Two new blaNDM-1 alleles that have polymorphisms in the signal peptide; NDM-1(P9R), a proline to arginine substitution, and NDM-2, a proline to alanine substitution (P28A) were studied. Here, we show that both the P9R and P28A substitutions improve secretion compared to NDM-1 and display higher resistance to some ß-lactam antibiotics. Mass spectrometry analysis of these purified NDM proteins showed that the P28A mutation in NDM-2 creates new signal peptide cleavage sites at positions 27 and 28. For NDM-1, we detected a signal peptide cleavage site between L21/M22 of the precursor protein. We find no evidence that NDM-1 is a lipoprotein, as has been reported elsewhere. In addition, expression of NDM-2 improves the fitness of E. coli, compared to NDM-1, in the absence of antibiotic selection. This study shows how optimization of the secretion efficiency of NDM-1 leads to increased resistance and increased fitness.

Effects of increased scrotal temperature on semen quality and seminal plasma proteins in Brahman bulls.

Environmental temperature has effects on sperm quality with differences in susceptibility between cattle subspecies and breeds, but very little is known about the seminal plasma protein (SPP) changes resulting from testicular heat stress. Scrotal insulation (SI) for 48 hr was applied to Brahman (Bos indicus) bulls. Semen was collected at 3-day intervals from before, until 74 days post-SI. The changes in sperm morphology and motility following SI were comparable to previously reported and differences were detected in measures of sperm chromatin conformation as early as 8 days post-SI. New proteins spots, in the SPP two-dimensional (2-D) gels, were apparent when comparing pre-SI with 74 days post-SI, and SPP identified as associated with mechanisms of cellular repair and protection. Similar trends between 2-D gel and Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) data was observed, with SWATH-MS able to quantify individual SPP that otherwise were not resolved on 2-D gel. The SPP assessment at peak sperm damage (21-24 days) showed a significant difference in 29 SPP (adjusted p < .05), and identified six proteins with change in abundance in the SI group. In conclusion both spermatozoa and SPP composition of bulls are susceptible to temperature change incurred by SI, and SPP markers for testicular heat insults may be detected.

Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families.

Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted "S"-shaped conformation when bound to A. thaliana AHAS (AtAHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors.

Assessing the Binding of Venoms from Aquatic Elapids to the Nicotinic Acetylcholine Receptor Orthosteric Site of Different Prey Models.

The evolution of an aquatic lifestyle from land dwelling venomous elapids is a radical ecological modification, bringing about many evolutionary changes from morphology to diet. Diet is an important ecological facet which can play a key role in regulating functional traits such as venom composition and prey-specific targeting of venom. In addition to predating upon novel prey (e.g., fish, fish eggs and invertebrates), the venoms of aquatic elapids also face the challenge of increased prey-escape potential in the aquatic environment. Thus, despite the independent radiation into an aquatic niche on four separate occasions, the venoms of aquatic elapids are evolving under convergent selection pressures. Utilising a biolayer interferometry binding assay, this study set out to elucidate whether crude venoms from representative aquatic elapids were target-specific to the orthosteric site of postsynaptic nicotinic acetylcholine receptor mimotopes of fish compared to other terrestrial prey types. Representatives of the four aquatic lineages were: aquatic coral snakes representative was Micrurus surinamensis;, sea kraits representative was Laticauda colubrina; sea snakes representatives were two Aipysurus spp. and eight Hydrophis spp; and water cobras representative was Naja annulata. No prey-specific differences in crude venom binding were observed from any species tested, except for Aipysurus laevis, which showed slight evidence of prey-potency differences. For Hydrophis caerulescens, H. peronii, H. schistosus and M. surinamensis, there was a lack of binding to the orthosteric site of any target lineage. Subsequent testing on the in vitro chick-biventer cervicis muscle preparation suggested that, while the venoms of these species bound postsynaptically, they bound to allosteric sites rather than orthosteric. Allosteric binding is potentially a weaker but faster-acting form of neurotoxicity and we hypothesise that the switch to allosteric binding is likely due to selection pressures related to prey-escape potential. This research has potentially opened up the possibility of a new functional class of toxins which have never been assessed previously while shedding light on the selection pressures shaping venom evolution.

Process Proteomics of Beer Reveals a Dynamic Proteome with Extensive Modifications.

Modern beer production is a complex industrial process. However, some of its biochemical details remain unclear. Using mass spectrometry proteomics, we have performed a global untargeted analysis of the proteins present across time during nanoscale beer production. Samples included sweet wort produced by a high temperature infusion mash, hopped wort, and bright beer. This analysis identified over 200 unique proteins from barley and yeast, emphasizing the complexity of the process and product. We then used data independent SWATH-MS to quantitatively compare the relative abundance of these proteins throughout the process. This identified large and significant changes in the proteome at each process step. These changes described enrichment of proteins by their biophysical properties, and identified the appearance of dominant yeast proteins during fermentation. Altered levels of malt modification also quantitatively changed the proteomes throughout the process. Detailed inspection of the proteomic data revealed that many proteins were modified by protease digestion, glycation, or oxidation during the processing steps. This work demonstrates the opportunities offered by modern mass spectrometry proteomics in understanding the ancient process of beer production.

Three-Finger Toxin Diversification in the Venoms of Cat-Eye Snakes (Colubridae: Boiga).

The Asian genus Boiga (Colubridae) is among the better studied non-front-fanged snake lineages, because their bites have minor, but noticeable, effects on humans. Furthermore, B. irregularis has gained worldwide notoriety for successfully invading Guam and other nearby islands with drastic impacts on the local bird populations. One of the factors thought to allow B. irregularis to become such a noxious pest is irditoxin, a dimeric neurotoxin composed of two three-finger toxins (3FTx) joined by a covalent bond between two newly evolved cysteines. Irditoxin is highly toxic to diapsid (birds and reptiles) prey, but roughly 1000 × less potent to synapsids (mammals). Venom plays an important role in the ecology of all species of Boiga, but it remains unknown if any species besides B. irregularis produce irditoxin-like dimeric toxins. In this study, we use transcriptomic analyses of venom glands from five species [B. cynodon, B. dendrophila dendrophila, B. d. gemmicincta, B. irregularis (Brisbane population), B. irregularis (Sulawesi population), B. nigriceps, B. trigonata] and proteomic analyses of B. d. dendrophila and a representative of the sister genus Toxicodryas blandingii to investigate the evolutionary history of 3FTx within Boiga and its close relative. We found that 92.5% of Boiga 3FTx belong to a single clade which we refer to as denmotoxin-like because of the close relation between these toxins and the monomeric denmotoxin according to phylogenetic, sequence clustering, and protein similarity network analyses. We show for the first time that species beyond B. irregularis secrete 3FTx with additional cysteines in the same position as both the A and B subunits of irditoxin. Transcripts with the characteristic mutations are found in B. d. dendrophila, B. d. gemmicincta, B. irregularis (Brisbane population), B. irregularis (Sulawesi population), and B. nigriceps. These results are confirmed by proteomic analyses that show direct evidence of dimerization within the venom of B. d. dendrophila, but not T. blandingii. Our results also suggest the possibility of novel dimeric toxins in other genera such as Telescopus and Trimorphodon. All together, this suggests that the origin of these peculiar 3FTx is far earlier than was appreciated and their evolutionary history has been complex.

Mechanisms of Persistence of the Ammonia-Oxidizing Bacteria Nitrosomonas to the Biocide Free Nitrous Acid.

Free nitrous acid (FNA) exerts a broad range of antimicrobial effects on bacteria, although susceptibility varies considerably among microorganisms. Among nitrifiers found in activated sludge of wastewater treatment processes (WWTPs), nitrite-oxidizing bacteria (NOB) are more susceptible to FNA compared to ammonia-oxidizing bacteria (AOB). This selective inhibition of NOB over AOB in WWTPs bypasses nitrate production and improves the efficiency and costs of the nitrogen removal process in both the activated sludge and anaerobic ammonium oxidation (Anammox) system. However, the molecular mechanisms governing this atypical tolerance of AOB to FNA have yet to be understood. Herein we investigate the varying effects of the antimicrobial FNA on activated sludge containing AOB and NOB using an integrated metagenomics and label-free quantitative sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) metaproteomic approach. The Nitrosomonas genus of AOB, on exposure to FNA, maintains internal homeostasis by upregulating a number of known oxidative stress enzymes, such as pteridine reductase and dihydrolipoyl dehydrogenase. Denitrifying enzymes were upregulated on exposure to FNA, suggesting the detoxification of nitrite to nitric oxide. Interestingly, proteins involved in stress response mechanisms, such as DNA and protein repair enzymes, phage prevention proteins, and iron transport proteins, were upregulated on exposure to FNA. In addition enzymes involved in energy generation were also upregulated on exposure to FNA. The total proteins specifically derived from the NOB genus Nitrobacter was low and, as such, did not allow for the elucidation of the response mechanism to FNA exposure. These findings give us an understanding of the adaptive mechanisms of tolerance within the AOB Nitrosomonas to the biocidal agent FNA.

Antifungal benzo[b]thiophene 1,1-dioxide IMPDH inhibitors exhibit pan-assay interference (PAINS) profiles.

Fungi cause serious life-threatening infections in immunocompromised individuals and current treatments are now complicated by toxicity issues and the emergence of drug resistant strains. Consequently, there is a need for development of new antifungal drugs. Inosine monophosphate dehydrogenase (IMPDH), a key component of the de novo purine biosynthetic pathway, is essential for growth and virulence of fungi and is a potential drug target. In this study, a high-throughput screen of 114,000 drug-like compounds against Cryptococcus neoformans IMPDH was performed. We identified three 3-((5-substituted)-1,3,4-oxadiazol-2-yl)thio benzo[b]thiophene 1,1-dioxides that inhibited Cryptococcus IMPDH and also possessed whole cell antifungal activity. Analogs were synthesized to explore the SAR of these hits. Modification of the fifth substituent on the 1,3,4-oxadiazole ring yielded compounds with nanomolar in vitro activity, but with associated cytotoxicity. In contrast, two analogs generated by substituting the 1,3,4-oxadiazole ring with imidazole and 1,2,4-triazole gave reduced IMPDH inhibition in vitro, but were not cytotoxic. During enzyme kinetic studies in the presence of DTT, nucleophilic attack of a free thiol occurred with the benzo[b]thiophene 1,1-dioxide. Two representative compounds with substitution at the 5 position of the 1,3,4-oxadiazole ring, showed mixed inhibition in the absence of DTT. Incubation of these compounds with Cryptococcus IMPDH followed by mass spectrometry analysis showed non-specific and covalent binding with IMPDH at multiple cysteine residues. These results support recent reports that the benzo[b]thiophene 1,1-dioxides moiety as PAINS (pan-assay interference compounds) contributor.

Insights into the interfacial structure-function of poly(ethylene glycol)-decorated peptide-stabilised nanoscale emulsions.

The interfacial properties of nanoscale materials have profound influence on biodistribution and stability as well as the effectiveness of sophisticated surface-encoded properties such as active targeting to cell surface receptors. Tailorable nanocarrier emulsions (TNEs) are a novel class of oil-in-water emulsions stabilised by molecularly-engineered biosurfactants that permit single-pot stepwise surface modification with related polypeptides that may be chemically conjugated or genetically fused to biofunctional moieties. We have probed the structure and function of poly(ethylene glycol) (PEG) used to decorate TNEs in this way. The molecular weight of PEG decorating TNEs has considerable impact on the ζ-potential of the emulsion particles, related to differential interfacial thickness of the PEG layer as determined by X-ray reflectometry. By co-modifying TNEs with an antibody fragment, we show that the molecular weight and density of PEG governs the competing parameters of accessibility of the targeting moiety and of shielding the interface from non-specific interactions with the environment. The fundamental understanding of the molecular details of the PEG layer that we present provides valuable insights into the structure-function relationship for soft nanomaterial interfaces. This work therefore paves the way for further rational design of TNEs and other nanocarriers that must interact with their environment in controlled and predictable ways.

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity.

Protein glycosylation is a critical post-translational modification that regulates the structure, stability, and function of many proteins. Mass spectrometry is currently the preferred method for qualitative and quantitative characterization of glycosylation. However, the inherent heterogeneity of glycosylation makes its analysis difficult. Quantification of glycosylation occupancy, or macroheterogeneity, has proven to be especially challenging. Here, we used a variation of high-resolution multiple reaction monitoring (MRM(HR)) or pseudo-MRM for targeted data-independent acquisition that we term SWAT (sequential window acquisition of targeted fragment ions). We compared the analytical performance of SWATH (sequential window acquisition of all theoretical fragment ions), SWAT, and SRM (selected reaction monitoring) using a suite of synthetic peptides spiked at various concentrations into a complex yeast tryptic digest sample. SWAT provided superior analytical performance to SWATH in a targeted approach. We then used SWAT to measure site-specific N-glycosylation occupancy in cell wall glycoproteins from yeast with defects in the glycosylation biosynthetic machinery. SWAT provided robust measurement of occupancy at more N-glycosylation sites and with higher precision than SWATH, allowing identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of oligosaccharyltransferase.

Commercial Herbicides Can Trigger the Oxidative Inactivation of Acetohydroxyacid Synthase.

Acetohydroxyacid synthase (AHAS) inhibitors are highly successful commercial herbicides. New kinetic data show that the binding of these compounds leads to reversible accumulative inhibition of AHAS. Crystallographic data (to a resolution of 2.17 Å) for an AHAS-herbicide complex shows that closure of the active site occurs when the herbicidal inhibitor binds, thus preventing exchange with solvent. This feature combined with new kinetic data shows that molecular oxygen promotes an accumulative inhibition leading to the conclusion that the exceptional potency of these herbicides is augmented by subversion of an inherent oxygenase side reaction. The reactive oxygen species produced by this reaction are trapped in the active site, triggering oxidation reactions that ultimately lead to the alteration of the redox state of the cofactor flavin adenine dinucleotide (FAD), a feature that accounts for the observed reversible accumulative inhibition.

High-antibody-producing Chinese hamster ovary cells up-regulate intracellular protein transport and glutathione synthesis.

Chinese hamster ovary (CHO) cells are the preferred production host for therapeutic monoclonal antibodies (mAb) due to their ability to perform post-translational modifications and their successful approval history. The completion of the genome sequence for CHO cells has reignited interest in using quantitative proteomics to identify markers of good production lines. Here we applied two different proteomic techniques, iTRAQ and SWATH, for the identification of expression differences between a high- and low-antibody-producing CHO cell lines derived from the same transfection. More than 2000 proteins were quantified with 70 of them classified as differentially expressed in both techniques. Two biological processes were identified as differentially regulated by both methods: up-regulation of glutathione biosynthesis and down-regulation of DNA replication. Metabolomic analysis confirmed that the high producing cell line displayed higher intracellular levels of glutathione. SWATH further identified up-regulation of actin filament processes and intracellular transport and down regulation of several growth-related processes. These processes may be important for conferring high mAb production and as such are promising candidates for targeted engineering of high-expression cell lines.

Sensitive detection of human insulin using a designed combined pore approach.

A unique combined pore approach to the sensitive detection of human insulin is developed. Through a systematic study to understand the impact of pore size and surface chemistry of nanoporous materials on their enrichment and purification performance, the advantages of selected porous materials are integrated to enhance detection sensitivity in a unified two-step process. In the first purification step, a rationally designed large pore material (ca. 100 nm in diameter) is chosen to repel the interferences from nontarget molecules. In the second enrichment step, a hydrophobically modified mesoporous material with a pore size of 5 nm is selected to enrich insulin molecules. A low detection limit of 0.05 ng mL(-1) in artificial urine is achieved by this advanced approach, similar to most antibody-based analysis protocols. This designer approach is efficient and low cost, and thus has great potential in the sensitive detection of biomolecules in complex biological systems.

Direct involvement of the TEN domain at the active site of human telomerase.

Telomerase is a ribonucleoprotein that adds DNA to the ends of chromosomes. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is important for activity and processivity. Here we describe a mutation in the TEN domain of human TERT that results in a greatly increased primer K(d), supporting a role for the TEN domain in DNA affinity. Measurement of enzyme kinetic parameters has revealed that this mutant enzyme is also defective in dNTP polymerization, particularly while copying position 51 of the RNA template. The catalytic defect is independent of the presence of binding interactions at the 5'-region of the DNA primer, and is not a defect in translocation rate. These data suggest that the TEN domain is involved in conformational changes required to position the 3'-end of the primer in the active site during nucleotide addition, a function which is distinct from the role of the TEN domain in providing DNA binding affinity.