Ammonium Bicarbonate Buffer Research Articles

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.

The GlycoFilter: A Simple and Comprehensive Sample Preparation Platform for Proteomics, N-Glycomics and Glycosylation Site Assignment

Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either N-glycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of N-glycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed de-N-glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including de-N-glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin, and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their nonsugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, glycoprotein quantification based exclusively on deglycosylated peptides may lead to inaccurate quantification.

Identification of the long-acting erythropoiesis-stimulating agent darbepoetin alfa in human urine by liquid chromatography–tandem mass spectrometry

The misuse of recombinant human erythropoietin (rhEPO) increases the proliferation/production of erythrocytes, which enhance oxygen transport capacities, and has grave consequences with respect to human health and fairness in sports. For sports drug testing, the current analytical methods for rhEPOs are mainly gel electrophoretic methods, such as isoelectric focusing-polyacrylamide gel electrophoresis. Mass spectrometry is fundamentally necessary for the reliable identification of rhEPOs in doping control. In this study, a high-sensitivity and high-throughput mass spectrometric qualitative detection method for darbepoetin alfa in human urine was established by a bottom-up approach. The novel method involves the immunopurification of human urine (10 mL), protease digestion with endoproteinase Glu-C (V8-protease) in an ammonium bicarbonate buffer (pH 7.8) and ultra-performance liquid chromatography using a charged surface hybrid C18 column coupled with electrospray-ionisation high-sensitivity tandem mass spectrometry for improved selectivity of the target molecules. The specific fragment digested from darbepoetin alfa was (90)TLQLHVDKAVSGLRSLTTLLRALGAQKE(117) (V11). The lower limit of detection of urinary darbepoetin alfa was 1.2 pg/mL. The limit of detection for the confirmation analysis was estimated to be 5 pg/mL. The developed method allows high-throughput confirmation analysis, namely 6 h for sample preparation and an analytical run time of only 10 min per sample; this high-throughput method dramatically decreases the workload in the laboratory. Darbepoetin alfa could be identified in human urine collected after the intravenous administration of 15 μg darbepoetin alfa (n = 3). This mass spectrometric method is an innovative and powerful tool for detecting darbepoetin alfa in human urine for doping control testing.

Preparation of Boronic Acid-Functionalized Mesoporous Nanomaterial and Its Application in Enrichment of Glycopeptides

A boronic acid-functionalized mesoporous nanomaterial was prepared by using a two-step post-grafting method. Firstly, the 3-glycidyloxypropyltrimethoxysilane (GLYMO) and 3-aminophenylboronic acid monohydrate (APB) were re- acted in oxyhydrogen sodium solution (pH 9.18) to prepare boronic-acid bonded GLYMO (denoted as GLYMO-APB). Sec- ondly, the MCM-41 was added into the prepared GLYMO-APB solution to prepare the final product of boronic acid func- tionalized MCM-41 (denoted as MCM-41-GLYMO-APB). This as-prepared material was characterized by FT-IR and the results showed that the boronic acid groups were successfully grafted to MCM-41. Glycopeptides can be enriched with high selectivity and high efficiency based on the formation of a cyclic diester between boronic hydroxyl and cis-diol on the glycan chain. The conditions of enriching glycopeptides in mixed digests of standard proteins by MCM-41-GLYMO-APB were compared and optimized. The incubation time, incubation solutions, washing ways and eluents used in the enrichment of glycopeptides were investigated. When using the ammonium bicarbonate buffer (100 mmol/L, pH 8.0) as the incubation so- lution and incubated for 1 h, the maximum number of enriched glycopeptides from horseradish peroxidase (HRP) digests could be obtained. After enrichment, washing the prepared material with the incubation solution twice for 5 min, nonglyco- peptides interference can be in a large extent removed. The enriched glycopeptides binding to the surface of MCM-41-GLYMO-APB could be released when using 1% trifluoroacetic acid containing 2,5-dihydroxybenzoic acid as the eluent, the operating steps could be simplified when using this optimized eluent because there was no need of adding the MALDI matrix solution afterwards. Therefore, this optimized protocol provided a reference for the glycopeptide enrichment with boronic acid-functionalized materials, and it also laid the foundation for the research of utilizing boronic acid-based chemical method to the glycopeptide enrichment in biological samples. Meanwhile, the highly ordered hexagonal cylindrical mesoporous structure of MCM-41-GLYMO-APB provided the possibility of being used for the research of glycopeptidome in biological samples. Keywords glycoproteins; boronic acid-functionalized mesoporous nanomaterial; enrichment; mass spectrometry

Development and validation of an LC‐ESI‐MS/MS quantification method for a potentialγ‐aminobutyric acid transporter 3 (GAT3) marker and its application in preliminary MS binding assays

Binding assays for the γ-aminobutyric acid (GABA) transporter GAT3 can be assumed to significantly facilitate screening for respective inhibitors. As appropriate labeled ligands for this promising drug target are not available so far, we started efforts to set up mass spectrometry-based binding assays (MS binding assays), for which labeled markers are not required. Therefore, we developed a sensitive and rapid LC-ESI-MS/MS quantification method for DDPM-1007 {(RS)-1-[4,4,4-Tris(4-methoxyphenyl)but-2-en-1-yl]piperidine-3-carboxylic acid}, one of the most potent GAT3 inhibitors yet known, as a potential GAT3 marker. Using a 50 × 2 mm C(8) column in combination with a mobile phase composed of 10 mM ammonium bicarbonate buffer pH 8.0 and acetonitrile (60:40, v/v) at a flow rate of 450 μL/min DDPM-1007 could be analyzed in the positive multiple reaction monitoring mode [(m/z) 502.5 → 265.4] within a chromatographic cycle time of 3 min. Deuterated DDPM-1007 [((2)H(9))DDPM-1007] was synthesized and employed as internal standard. This way DDPM-1007 could be quantified in a range from 100 pM to 10 nM in the matrix resulting from respective binding experiments without any sample preparation. The established quantification method met the requirements of the FDA guidance for bioanalytical method validation concerning linearity and intra- and inter-batch accuracy. Based on this LC-ESI-MS/MS quantification preliminary MS binding assays employing membrane preparations obtained from a stably GAT3 expressing HEK293 cell line and DDPM-1007 as nonlabeled GAT3 marker could be performed. In these experiments specific binding of DDPM-1007 at GAT3 could be unambiguously detected. Additionally, the established LC-MS method provides a suitable analytical tool for further pharmacokinetic characterization of DDPM-1007, as exemplified for its logD determination.

Extraction Of House Dust Mite Allergen, Der P1, From Dust

A recent article in this journal found that the type of extraction buffer did not affect measured levels of the major group one allergen (Der p 1) from the house dust mite, Dermatophagoides pteronyssinus, in household dust samples (1). This differed from a previous study where about twice the levels of Der p 1 were found in borate-buffered saline extracts, compared to ammonium bicarbonate buffer or phosphate-buffered saline extracts, irrespective of extraction temperature or time of extraction (2). In discussing our previous study (2), the authors state that we did not use Tween-20 in our extraction buffers. Although our article did not make a mention of Tween-20, we in fact did use it in our extraction media. It was an oversight of us in that we did not mention this in our article. We further explored temperature and buffer effects on Der p 1 extraction in an external quality control program where six dust samples were sent to 23 laboratories worldwide (3). Results from this study again showed that type of extraction buffer and extraction temperature influenced Der p 1 measurements. The majority of the participant laboratories did use Tween-20 as a wetting agent in their extraction buffers. As pointed out by the authors, adding Tween-20 to extraction media results in higher endotoxin levels and is recommended by the European Committee for Standardisation. The authors call for further investigation on the effects of Tween-20 on extraction effi ciency of all indoor allergens from dust. However, the majority of indoor allergen analysis laboratories use ELISA or MARIATM kits from Indoor Biotechnologies where the recommended extraction medium is phosphate-buffered saline containing 0.02 % Tween-20. It would take much effort to study this systematically to obtain results with suffi cient internal and external validity.

Feasibility and application of an HPLC/UVD to determine dinotefuran and its shorter wavelength metabolites residues in melon with tandem mass confirmation

A new analytical method was developed for dinotefuran and its metabolites, MNG, UF, and DN, in melon using high-performance liquid chromatography (HPLC) coupled with an ultraviolet detector (UVD). Due to shorter wavelength, lower sensitivity to UV detection, and high water miscibility of some metabolites, QuEChERs acetate-buffered version was modified for extraction and purification. Mobile phases with different ion pairing or ionisation agents were tested in different reverse phase columns, and ammonium bicarbonate buffer was found as the best choice to increase the sensitivity of target analytes to the UV detector. After failure of dispersive SPE clean-up with primary secondary amine, different solid phase extraction cartridges (SPE) were used to check the protecting capability of analytes against matrix interference. Finally, samples were extracted with a simple and rapid method using acetonitrile and salts, and purified through C18SPE. The method was validated at two spiking levels (three replicates for each) in the matrix. Good recoveries were observed for all of the analytes and ranged between 70.6% and 93.5%, with relative standard deviations of less than 10%. Calibration curves were linear over the calibration ranges for all the analytes with r2⩾0.998. Limits of detection ranged from 0.02 to 0.05mgkg−1, whereas limits of quantitation ranged from 0.06 to 0.16mgkg−1 for dinotefuran and its metabolites. The method was successfully applied to real samples, where dinotefuran and UF residues were found in the field-incurred melon samples. Residues were confirmed via LC–tandem mass spectrometry (LC–MS/MS) in positive-ion electrospray ionisation (ESI+) mode.

LmrTX, a basic PLA2 (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity

A basic phospholipase A2 (LmrTX) isoform was isolated from Lachesis muta rhombeata snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-5 Discovery® Bio Wide column. From liquid chromatography–electrospray ionization/mass spectrometry, the molecular mass of LmrTX was measured as 14.277.50 Da. The amino acid sequence showed a high degree of homology between PLA2 LmrTX from L. muta rhombeata and other PLA2 from snake venoms, like CB1 and CB2 from Crotalus durissus terrificus; LmTX-I and LmTX-II from Lachesis muta muta. LmrTX had PLA2 activity in the presence of a synthetic substrate and alkylation of histidine residues significantly inhibited (P < 0.05) the enzymatic activity of LmrTX and its anticoagulant and antithrombotic activity. In this study, we examined the ability of the LmrTX in altering thrombus formation in living mouse, using a photochemically induced arterial thrombosis model. The control animals that did not receive protein injection showed a normal occlusion time, which was around 57 ± 7.8 min. LmrTX, the PLA2 from L. muta rhombeata venom, caused a change in the occlusion time to 99 ± 10 min with doses of 7.5 μg/mice. Additionally, LmrTX showed the anticoagulant activity in vitro and ex vivo and prolonging the time aggregation in wash platelet induced by ADP and Thrombin.

Development and Validation of Selective High-Performance Liquid Chromatographic Method Using Photodiode Array Detection for Estimation of Aconitine in Polyherbal Ayurvedic Taila Preparations

A simple, sensitive, and selective high-performance liquid chromatographic (HPLC) method has been developed and validated for the analysis of aconitine in marketed ayurvedic taila (oil) formulations containing roots of Aconitum chasmanthum. Chromatography of methanolic extracts of these formulations was performed on C18 (5 μm × 25 cm × 4.6 mm i.d.) column using isocratic mobile phase consisting of (65 : 35% v/v) acetonitrile and buffer solution (aqueous 0.01 M ammonium bicarbonate buffer, adjusted to pH 9.6 using 30% ammonia solution) at a flow rate of 1 mL/min and SPD-10 AVP photodiode array (PDA) UV-Visible detector. The analytical reference, aconitine, was quantified at 238 nm. The retention time of aconitine was about 42.54 min. The linear regression analysis data for the calibration plot showed a good linear relationship with correlation coefficient of 0.9989 in the concentration range of 15 to 90 μg/mL for aconitine with respect to peak area. The limit of detection and limit of quantitation values were found to be 0.03 μg/mL and 0.1 μg/mL respectively. Repeatability of the method was found to be 0.551–1.689 RSD. Recovery values from 97.75 to 99.91% indicate excellent accuracy of the method. The developed HPLC method is accurate and precise and it can be successfully applied for the determination of aconitine in marketed ayurvedic oil formulations containing Aconitum chasmanthum.

Studies of Histidine As a Suitable Isoelectric Buffer for Tryptic Digestion and Isoelectric Trapping Fractionation Followed by Capillary Electrophoresis–Mass Spectrometry for Proteomic Analysis

The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using "membrane separated wells for isoelectric focusing and trapping" (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers. In addition, this sample preparation method and MSWIFT separations have been coupled with capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as an alternative separation approach for proteomic studies. Tryptic peptides of ribosomal proteins in histidine are fractionated using MSWIFT followed by CE-MALDI-MS, which further illustrates the ability to couple fractions from a pI based separation device to CE-MS. Specifically, two-dimensional CE-MS plots provide a direct correlation between the numbers of basic residues within the peptide sequence displayed in charge-state trend lines. Combining MSWIFT and CE-MS provides added information regarding peptide sequence, specifically pI and in-solution charge state. Post-translational modifications can also be identified using this method.

Comparative analysis of aromatic diisocyanate conjugation to human albumin utilizing multiplexed tandem mass spectrometry

Diisocyanates are industrially important chemicals that serve as polymerizing agents in a variety of polyurethane products. In addition to their many industrial uses, diisocyanates have been implicated as causative agents of occupational allergic respiratory disease, although the specific mechanism(s) by which these diseases occur remains unknown. In this study the sites of conjugation of the two most industrially important monomeric diisocyanates, methylene diphenyl diisocyanate (MDI) and toluene diisocyanate (TDI) on human serum albumin are identified utilizing multiplexed tandem mass spectrometry on a quadrupole time-of-flight mass spectrometer. Analysis of human albumin reacted with MDI and TDI over the range of 1:1–40:1 (isocyanate:protein) mol ratio reveals that MDI and TDI react with a maximum of 20 and 37 residues, respectively. Conjugation of diisocyanates to albumin proceeds in a concentration-dependant manner with MDI and TDI reacting at a preferred subset of 5 and 10 residues, respectively, in the limiting case of a 1:1 mol ratio. MDI reacts at fewer residues than does TDI, and is not observed to react with any residues exclusive of TDI. These results cannot be explained on the basis of simple sterics or hydrophobicity, but rather on the basis of increased reactivity of one TDI isocyanate moiety due to electron withdrawing character of the second isocyanate moiety. Furthermore, reaction of diisocyanates with albumin in a phosphate buffered saline (PBS) solution provides three additional reactive sites that are not observed in ammonium bicarbonate buffer. Two lysine residues, Lys 199 and Lys 525, are observed to be reactive to both diisocyanates at all concentrations and in all solvent systems employed in this study. This study presents a comprehensive conjugation map of MDI and TDI on human albumin. The results suggest that several albumin residues are reactive toward both MDI and TDI and may be useful for biomonitoring of diisocyanate exposures.

A novel and non-destructive approach for ZooMS analysis: ammonium bicarbonate buffer extraction

Bone collagen is found throughout most of the archaeological record. Under experimental conditions, collagen is apparently preserved as an intact molecule, with amino acid compositions and isotopic profiles only changing when almost all of the protein is lost. The ubiquity of collagen in archaeological bone has lead to the development of the use of collagen peptide mass fingerprints for the identification of bone fragments—Zooarchaeology by Mass Spectrometry (ZooMS). We report a novel, but a simple method for the partial extraction of collagen for ZooMS that uses ammonium bicarbonate buffer but avoids demineralisation. We compared conventional acid demineralisation with ammonium bicarbonate buffer extraction to test ZooMS in a range of modern and archaeological bone samples. The sensitivity of the current generation of mass spectrometers is high enough for the non-destructive buffer method to extract sufficient collagen for ZooMS. We envisage that a particular advantage of this method is that it leaves worked bone artefacts effectively undamaged post-treatment, suitable for subsequent analysis or museum storage or display. Furthermore, it may have potential as a screening tool to aid curators in the selection of material for more advanced molecular analysis—such as DNA sequencing.

Development and validation of a rapid LC-ESI-MS/MS method for quantification of fluoxetine and its application to MS binding assays

In the present study, a rapid and sensitive LC-ESI-MS/MS method for quantification of (S)-fluoxetine as a native marker in mass spectrometry (MS) binding assays addressing the human serotonin transporter (hSERT) was developed and validated. The concept of MS binding assays based on mass spectrometric quantification of a nonlabeled marker recently introduced by us represents a promising alternative to conventional radioligand binding without the drawbacks inherently connected with radioisotope labeling. For high-performance liquid chromatography (HPLC), a 20 × 2-mm RP-18 column with a mobile phase composed of acetonitrile and ammonium bicarbonate buffer (5 mmol L(-1), pH 9.5) at a ratio of 80:20 (v/v) and a flow rate of 800 μL min(-1) in an isocratic mode were used, resulting in a chromatographic cycle time of 60 s. Employing [(2)H(5)]fluoxetine as internal standard enabled ESI-MS/MS quantification of (S)-fluoxetine between 3 nmol L(-1) and 50 pmol L(-1) (LLOQ) in matrix obtained from binding experiments without the need of any sample preparation. Validation of the method showed that linearity, intra-, and inter-batch accuracy as well as precision meet the requirements of the FDA guidance for bioanalytical method validation. Considering sensitivity and speed, the established method is clearly superior to those published for biological matrices so far. Furthermore, the method was transferred to other RP-18 columns of different lengths and respective validation experiments demonstrated its versatility and chromatographic robustness. Finally, the newly developed method was successfully applied to MS binding assays for hSERT. The affinity determined for (S)-fluoxetine in saturation experiments was in good agreement with literature data obtained in respective radioligand binding assays.

Ultrasonic Enhanced Applications in Proteomics Workflows: single probe versus multiprobe.

Ultrasonic Enhanced Applications in Proteomics Workflows: single probe versus multiprobe.

Increased Selectivity, Analytical Precision, and Throughput in Targeted Proteomics

Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies.

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.

Global Proteomics Reveal an Atypical Strategy for Carbon/Nitrogen Assimilation by a Cyanobacterium Under Diverse Environmental Perturbations

Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis, are present in diverse ecological niches and play crucial roles in global carbon and nitrogen cycles. To proliferate in nature, cyanobacteria utilize a host of stress responses to accommodate periodic changes in environmental conditions. A detailed knowledge of the composition of, as well as the dynamic changes in, the proteome is necessary to gain fundamental insights into such stress responses. Toward this goal, we have performed a large-scale proteomic analysis of the widely studied model cyanobacterium Synechocystis sp. PCC 6803 under 33 different environmental conditions. The resulting high-quality dataset consists of 22,318 unique peptides corresponding to 1955 proteins, a coverage of 53% of the predicted proteome. Quantitative determination of protein abundances has led to the identification of 1198 differentially regulated proteins. Notably, our analysis revealed that a common stress response under various environmental perturbations, irrespective of amplitude and duration, is the activation of atypical pathways for the acquisition of carbon and nitrogen from urea and arginine. In particular, arginine is catabolized via putrescine to produce succinate and glutamate, sources of carbon and nitrogen, respectively. This study provides the most comprehensive functional and quantitative analysis of the Synechocystis proteome to date, and shows that a significant stress response of cyanobacteria involves an uncommon mode of acquisition of carbon and nitrogen.

Successive multiple ionic polymer layer coated capillaries in the separation of proteins – Recombinant allergen variants as a case study

A successive multiple ionic polymer layer (SMIL) coating consisting of two pairs of poly(diallyldimethylammonium chloride) and dextran sulfate (DS) layers was applied for the separation of recombinant products of the major birch pollen allergen Betula verrucosa (Bet v 1a). The combination with volatile ammonium bicarbonate buffer at pH 6.70 offers the possibility for future MS hyphenation. The negative net charge of allergens required DS as terminal SMIL layer. The EOF was accelerated from 3.17x10(-8) m(2) V(-1) s(-1) in uncoated to 4.52x10(-8) m(2) V(-1) s(-1) in SMIL capillaries. Fresh prepared SMIL capillaries showed slight EOF acceleration due to gradual re-organization of SMIL structure until stabilization was achieved. Dry storage of SMIL capillaries prevented fluctuations in EOF and migration times and improved coating durability. However, the gradual reconstitution of entangled SMIL layers affected efficiency, but was cured by a 10 mmol/L NaOH rinsing step. Durability of SMIL capillaries in MS-applicable dimension was confirmed for > 70 runs and in total 42 h of voltage application with average intra-day precision of 0.22 and 0.79% and inter-day-precision of 0.91 and 1.17% for migration times of EOF and Bet v 1a, respectively. Final SMIL coating allowed for the separation of Bet v 1a, a hypoallergenic isoform and carbamylated variants with 150,000-685,000 plates.

Lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects

Caveolins form plasmalemnal invaginated caveolae. They also locate around intracellular lipid droplets but their role in this location remains unclear. By studying primary adipocytes that highly express caveolin-1, we characterized the impact of caveolin-1 deficiency on lipid droplet proteome and lipidome. We identified several missing proteins on the lipid droplet surface of caveolin-deficient adipocytes and showed that the caveolin-1 lipid droplet pool is organized as multi-protein complexes containing cavin-1, with similar dynamics as those found in caveolae. On the lipid side, caveolin deficiency did not qualitatively alter neutral lipids in lipid droplet, but significantly reduced the relative abundance of surface phospholipid species: phosphatidylserine and lysophospholipids. Caveolin-deficient adipocytes can form only small lipid droplets, suggesting that the caveolin-lipid droplet pool might be involved in lipid droplet size regulation. Accordingly, we show that caveolin-1 concentration on adipocyte lipid droplets positively correlated with lipid droplet size in obese rodent models and human adipocytes. Moreover, rescue experiments by caveolin- green fluorescent protein in caveolin-deficient cells exposed to fatty acid overload demonstrated that caveolin-coated lipid droplets were able to grow larger than caveolin-devoid lipid droplets. Altogether, these data demonstrate that the lipid droplet-caveolin pool impacts on phospholipid and protein surface composition of lipid droplets and suggest a functional role on lipid droplet expandability.

Effect of thermochemical pretreatment on sewage sludge and its impact on carboxylic acids production

This paper investigates the potential of converting sewage sludge into a useful product, namely carboxylic acids. To potentially enhance acid yields, the effect of pretreatment using 0.3 g lime/g dry biomass and water at 100 °C for 10–240 min was studied. The pretreated sludges were anaerobically fermented to mixed-acids using a mixed culture of microorganisms; methanogens were suppressed using iodoform. Batch fermentations were performed at 55 °C using ammonium bicarbonate buffer. The first batch experiments compared treated and untreated sludge as the only substrate. The second batch experiments used a mixture of sludge plus lime-treated bagasse (20:80 by weight). Analysis of liquor shows that the pretreatment were effective in solubilizing constituent compounds of sewage sludge. Nitrogen content and carboxylic acids increased with increasing pretreatment time. However, the soluble sugars peaked at 60 min, and then decreased with longer pretreatment time, showing that the solubilised sugars were undergoing intermolecular reactions, such as Maillard reactions. Fermentation experiments were a good indicator of the biodegradability of the pretreated sludges. Results clearly showed that lime-treating sludge, using even the minimum pretreatment time (10 min), negatively impacted acid production. The likely causes of this observation are attributed to the production of recalcitrant complexes and toxic compounds. Batch fermentation of untreated sludge yielded 0.34 g total acids/g VS fed, whereas sludge with 240-min lime pretreatment yielded only 0.20 g total acids/g VS fed. Co-fermentation of untreated sludge with pretreated bagasse gave a yield of 0.23 g total acids/g VS fed.