TOF Analysis Research Articles

Segmental copolymers of condensation polyesters and polylactide

L-Lactide (LA) polymerizations carried out in the presence of various types of polyesterodiols and polyesterocarbonatediols obtained from 1,4-butanediol, 1,3-propanediol and dimethyl esters of adipic, terephthalic and carbonic acids were studied. It was found, based on 1H NMR and MALDI ToF analysis of the chains microstructure that the reactions carried out in bulk at 190 °C in the presence of tin (II) 2-ethylhexanoate as a catalyst led to the formation of a mixture of polylactide (PLLA) and block copolymers of LA with the applied macrodiols with high yield. DSC studies of the obtained products indicate that the segments comprising of adipic and carbonic acid derivatives were fully miscible with PLLA causing the reduction of its glass transition temperature. The segments containing terephthalic acid monomeric units in its structure showed only slight miscibility with PLLA segments and the obtained products consist of two phases of glass transition temperatures close to those of PLLA and the macrodiol. These systems are characterized by better thermal stability and higher elasticity than PLLA.

Synthesis of functionalized polylactide by cationic activated monomer polymerization

Functionalized polylactides (PLAs) containing acryloyl, methacryloyl or propargyl end groups have been obtained by cationic ring-opening polymerization performed in the presence of appropriate alcohols (HEMA, HEA, propargyl alcohol) as initiators and triflic acid as a catalyst. 1H NMR, MALDI TOF and GPC analysis indicated almost quantitative initiation and confirmed the expected structure and molecular masses of the obtained PLAs. The conditions were found in which transesterification process (usually accompanying the cyclic esters propagation) can be avoided. PLAs functionalized with double bond were successfully homopolymerized and copolymerized with butyl acrylate in the presence of AIBN. PLA with triple bond at one chain end was effectively coupled with a model azide by 1,3-dipolar Huisgen cycloaddition (“click” reaction) in order to prove its ability to be further functionalized.

Health effects of environmental DDT exposure: An epidemiological and serum proteomic study

P offer a variety of benefits to the society such as increased crop production and decreased insect infestations. However, when used improperly, pesticides have the potential for causing harm. Approximately, 10,000-20,000 physiciandiagnosed pesticide poisonings occur each year among US agricultural workers. Although banned in the US in 1973, DDT, an organochlorine pesticide finds its way into the food chain as it is used in many parts of the world. The purpose of the study was to examine the health effects of DDT in a predominantly African American population who were exposed to highest levels of DDT in the US by consumption of DDTcontaminated fish. A cross-sectional study was conducted on 234 African Americans. Logistic Regression was used to calculate risk of morbidity from consumption of DDTcontaminated fish. Serum proteomic study included 21 breast cancer cases and 29 controls. Protein expression data was obtained by 2D DIGE analysis. Differentially expressed DIGE spots were excised for protein identification. Spots were digested with trypsin and the peptides were identified by QTOF LC/MS/MS or MALDI TOF analysis. Results: A non-significant increase in risk for hypertension in those who consumed DDT contaminated fish was observed (OR 1.672; 95% CI 0.324-8.628). Serum proteomic study yielded significantly increased levels of fibrinogen gamma in exposed individuals. Gamma fibrinogen is associated with increased risk of coronary artery disease. Future efforts will focus on validation of gamma fibrinogen in DDT exposed individuals. Funded by Susan G. Komen for the cure.

Rapid discrimination of environmental Vibrio by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The aim of this study was to discriminate 30 Vibrio strains isolated from two wastewater treatment plants from Agadir, Morocco by two molecular typing methods, pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Out of the 30 strains of Vibrio examined in this study, 5 isolates could not be typed by PFGE and consistently appeared as a smear on the gel. In general, high genetic biodiversity among the Vibrio strains was found regardless to the isolation source. The results of MALDI TOF analysis show a high congruence of strain grouping demonstrating the accuracy and reliability of MALDI-TOF MS.

PROSE: Randomized proteomic stratified phase III study of second line erlotinib versus chemotherapy in patients with inoperable non–small cell lung cancer (NSCLC).

TPS214 Background: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI ToF MS) of NSCLC patients’ pretreatment sera or plasma was shown to predict survival after EGFR TKIs therapy (Taguchi et al. JNCI 2007). The proteomic classifier (VeriStrat, Biodesix Inc), based on 8 distinct m/z peaks, separates patients (pts) into 2 groups (VeriStrat good and poor), significantly different in terms of time to progression and overall survival when treated with EGFR TKIs but not chemotherapy (CT). Aim of the current study is to prospectively evaluate the predictive value of the proteomic classifier on outcome in advanced NSCLC pts, treated with either EGFR-TKIs or standard CT in second-line setting. Methods: PROSE is a phase III, prospective, double-blinded trial where pts with advanced NSCLC are randomized in a 1:1 ratio to receive erlotinib 150 mg daily or standard second-line CT (pemetrexed 500 mg/m2 3w or docetaxel 75 mg/m2 3w). Pts are stratified according to ECOG Performance Status, smoking history and proteomic profile (good vs poor) obtained from MALDI ToF analysis of pretreatment sera. To detect a different survival effect between erlotinib and CT in the 2 proteomic-classified groups, the study sample size was calculated based on: the established 65/35% ratio between VeriStrat good and VeriStrat poor-classified pts; the expected median survival of 7 months (mos) in patients treated with CT irrespectively of proteomic profile and of approximately 4.2 mos for VeriStrat poor (HR 1.7) and 9.9 mos for VeriStrat good (HR 0.7) pts treated with erlotinib. Up to now, 196 pts out of the 275 planned have been enrolled in 12 Italian centers. PROSE trial is the first study worldwide that prospectively applies proteomics into a clinical setting. If the predictive value of proteomic analysis were confirmed, PROSE study would open the possibility of a new, simple and reliable tool to select the best second-line treatment between EGFR TKIs or CT in NSCLC pts, who are either EGFR undetermined or wild type.

Purification and Identification of a Natural Lectin from the Seed of Peanut Arachis hypogaea

A natural lectin from the seed of peanut Arachis hypogaea was purified by singlestep affinity chromatography using galactoside-coupled agarose. The native molecular mass of purified A. hypogaea lectin (PN-L) was 29 kDa. The lectin PN-L was detected for agglutinating activity, glycoinhibiting action and thermostability. The influence of pH on those activities was also tested. The results showed that PN-L could not agglutinate three kinds of human erythrocytes. But it showed a strong affinity to human A, B and O erythrocytes (RBC) treated by neuraminidase. Agglutinating activity of PN-L to neuraminidase treated human O erythrocytes was inhibited by lactose, raffinose, melibiose and D-galactose. The agglutinating activity of peanut seed lectin was stable up to 55°C and at pH 5.0-11.0. The results of MALDI-TOF- TOF analysis indicated that the protein PN-L showed highly homology with the Peanut Lectin Chain A protein (gi|1942899).

Virulence Factors, Antimicrobial Resistance Genes and Protein Structure of Crohn's Disease-Derived E.Coli Strains Identified Using DNA Microarray and MALDI-TOF Analysis

Introduction: Intracellular E. coli isolates are found in up to 60% of gut biopsies from patients with Crohn's disease (CD). Although adherent invasive E.coli (AIEC) are described[1], other intracellular E. coli are frequently isolated but specific pathogenic determinants for these have not been identified by conventional techniques. DNA microarray and Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry enable rapid and accurate DNA and proteomic analysis of bacteria. Aims: To investigate for virulence and antimicrobial resistance (AMR) genes in, and analyse the structure of CD-related intracellular E.coli isolates. Methods: Seventeen E.coli strains were isolated using gentamicin protection assay from gut biopsies of 17 CD patients (ileal n=7, colonic n=10). DNA was extracted for analysis by DNA microarray of 392 virulence and AMR genes. DNA PCR was performed for an additional 18 virulence genes. For MALDI-TOF sample preparation, several colonies of each bacterial strain were spotted onto target plate and prepared as described for Bruker Biotyper MALDI TOF analysis. Ten replicates of the mass/charge spectra for each strain were compared to spectra from a bacterial database Results: Virulence factors were identified by DNA microarray in a number of E.coli strains including senB (encodes enterotoxin), Iha (encodes an adhesin) Sat (toxin of uropathogenic E.Coli), iron regulation genes and microcin synthesis genes. DNA PCR revealed additional virulence factors including ibeA (associated with E.coli invasion in neonatal meningitis) in 2 strains. A minority of strains were positive for AMR genes relating to beta-lactams, sulphonamides, tetracycline and streptomycin. MALDI-TOF analysis confirmed the bacteria as E.coli and clustered the Crohn's disease E.coli strains together and separate from other E.coli strains according to protein structure on the Bruker database. There was no clustering within CD strains in relation to site of disease or biopsy, or use of immunomodulation. Conclusion: Although CD appears to be at least in part a host immune disorder, pathogenic factors of various organisms may still play a role in pathogenesis. A number of genes encoding for virulence factors have been identified in a proportion of CD-related E.coli strains which warrant further investigation (eg whole genome sequencing and functional assays). Most strains had no antimicrobial resistance genes. Further MALDI-TOF comparison of CD-related bacterial structure with commensal and IBD-related bacteria may be revealing.

Purification, Biochemical Characterization, and Bioactive Properties of a Lectin Purified from the Seeds of White Tepary Bean (Phaseolus Acutifolius Variety Latifolius)

The present work shows the characterization of Phaseolus acutifolius variety latifolius, on which little research has been published, and provides detailed information on the corresponding lectin. This protein was purified from a semi-domesticated line of white tepary beans from Sonora, Mexico, by precipitation of the aqueous extract with ammonium sulfate, followed by affinity chromatography on an immobilized fetuin matrix. MALDI TOF analysis of Phaseolus acutifolius agglutinin (PAA) showed that this lectin is composed of monomers with molecular weights ranging between 28 and 31 kDa. At high salt concentrations, PAA forms a dimer of 63 kDa, but at low salt concentrations, the subunits form a tetramer. Analysis of PAA on 2D-PAGE showed that there are mainly three types of subunits with isoelectric points of 4.2, 4.4, and 4.5. The partial sequence obtained by LC/MS/MS of tryptic fragments from the PAA subunits showed 90–100% identity with subunits from genus Phaseolus lectins in previous reports. The tepary bean lectin showed lower hemagglutination activity than Phaseolus vulgaris hemagglutinin (PHA-E) toward trypsinized human A and O type erythrocytes. The hemagglutination activity was inhibited by N-glycans from glycoproteins. Affinity chromatography with the immobilized PAA showed a high affinity to glycopeptides from thyroglobulin, which also has N-glycans with a high content of N-acetylglucosamine. PAA showed less mitogenic activity toward human lymphocytes than PHA-L and Con A. The cytotoxicity of PAA was determined by employing three clones of the 3T3 cell line, demonstrating variability among the clones as follows: T4 (DI50 51.5 µg/mL); J20 (DI50 275 µg/mL), and N5 (DI50 72.5 µg/mL).

Isolation and physico-chemical characterisation of extracellular polymeric substances produced by the marine bacterium Vibrio parahaemolyticus

A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 Å, respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. 1HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CIxrd 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF–TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.

Laccase-catalyzed oxidative polymerization of aniline dimer (N-phenyl-1,4-phenylenediamine) in aqueous micellar solution of sodium dodecylbenzenesulfonate

Abstract We have studied oxidative polymerization of aniline dimer (N-phenyl-1,4-phenylenediamine) catalyzed by high-redox potential laccase isolated from the fungi Trametes hirsuta ( Wulfen ) Pilat CF-28. Enzymatic aniline dimer polymerization was performed in aqueous micellar solution of sodium dodecylbenzenesulfonate, the atmospheric oxygen serving as an oxidizer. The resultant dispersion was stable for at least 6 months. The products synthesized were characterized using Fourier transform infrared and UV–vis spectroscopies. MALDI TOF analysis has shown that aniline dimers polymerize to mainly form aniline oligomers with the m/z ratio up to 2180, which corresponds to a polymerization degree of 24 (in terms of aniline subunits). Enzymatically formed aniline oligomers consist for the most part of para-directed units in the form of emeraldine salt. The end product structure depends on the reaction medium pH. Transmission electron microscopy has revealed granular nanoparticles of the reaction product.

Elongation factor 1-alpha is released into the culture medium during growth of Giardia intestinalis trophozoites

The molecular pathogenesis of the intestinal parasite Giardia intestinalis is still not fully understood but excretory–secretory products have been suggested to be important during host–parasite interactions. Here we used SDS–PAGE gels and MALDI–TOF analysis to identify proteins released by Giardia trophozoites during in vitro growth. Serum proteins (mainly bovine serum albumin) in the growth medium, bind to the parasite surface and they are continuously released, which interfere with parasite secretome characterization. However, we identified two released Giardia proteins: elongation factor-1 alpha (EF-1α) and a 58kDa protein, identified as arginine deiminase (ADI). This is the first description of EF-1α as a released/secreted Giardia protein, whereas ADI has been identified in an earlier secretome study. Two genes encoding EF-1α were detected in the Giardia WB genome 35kbp apart with almost identical coding sequences but with different promoter and 3′ regions. Promoter luciferase-fusions showed that both genes are transcribed in trophozoites. The EF-1α protein localizes to the nuclear region in trophozoites but it relocalizes to the cytoplasm during host–cell interaction. Recombinant EF-1α is recognized by serum from giardiasis patients. Our results suggest that released EF-1α protein can be important during Giardia infections.

Enhancement in MALDI‐TOF MS analysis of the low molecular weight human serum proteome

The combination in advances in MALDI-TOF MS instrumentation(1) and serum sample preparation techniques,(2-4) has lead to the emergence of MALDI serum protein expression profiling as a promising tool for biomarker discovery.(5) However, three factors still pose significant challenges for MALDI profiling of serum: the limited mass window of MALDI, serum protein complexity,(6) and analytical reproducibility.(7, 8) MALDI has a small mass preference due to the ionization efficiency of intact protein molecules and the focusing of flight of ions towards the detector. In linear mode, TOF analyzers have limited sensitivity for masses above 20 kDa. To address this, we previously reported the enhancement of MALDI broad mass range detection of protein signals.(9) Spanning a 100 kDa mass range, we improved the sensitivity of detection by an order of magnitude through the combined optimization of sample preparation, instrument parameters and data processing procedures. The complexity of the blood proteome is very high, with protein concentrations differing by up to ten orders of magnitude.(10) This large dynamic range exceeds current proteomic analytical capabilities; thus analysis of easily prepared subproteomes of serum or plasma is essential. Here, we shift our focus back to the enhancement of MALDI analysis in the low mass regime. The low molecular weight (LMW) subcomponent of serum promises to be a rich source of undiscovered biomarkers, as biological processes give rise to a plethora of proteolytic protein fragments.(11) Currently, there is no consensus on what constitutes the mass limits of this derivative proteome (also termed peptidome). However, “<15 kDa” is often cited in the literature based on a serum MALDI study using 20 MWCO filters.(12) To date, small native protein/peptide mass measurements have been mainly conducted in reflectron mode (< 4 kDa),(13) and linear mode up to 10 kDa.(14) However, no systematic comparison has been described for a combination of preparation steps: spotting, filtering and matrix choice, to optimize the performance. A rigorous reproducibility study for different preparation strategies is likewise missing, which prevents extension of the technique to clinical proteomics applications. The purpose of this work was to provide such a systematic comparison, using rigorous performance metrics to optimize sample preparation for LMW serum proteome profiling by MALDI-TOF MS analysis up to 20 kDa. We explored a combination of MALDI sample preparation and spotting methods. Procedures that gave the best results included: 1) MALDI spotting with a thin layer technique using sinapinic acid on a ground steel plate, and 2) centrifugal ultrafiltration with 50000 MWCO filters in the presence of 2% TFA, followed by desalting with C3 magnetic beads. Ultrafiltration has been utilized previously to improve MALDI profiling of serum.(2, 13, 15-17) However, our approach extensively improves MALDI peak intensities of higher MW peptides and small proteins, thus increasing biomarker coverage in the 3-20 kDa range. Reproducibility studies of a protein standard and serum samples gave excellent results, all of which suggests that this procedure can be a useful tool for proteome profiling of the LMW fraction of serum.

Detection, characterization and identification of major constituents in Zhimu‐Baihe herb‐pair extract by fast high‐performance liquid chromatography and time‐of‐flight mass spectrometry through dynamic adjustment of fragmentor voltage

This work describes a novel methodology for unequivocal identification of chemical constituents in Zhimu-Baihe herb-pair (ZMBHHP). Compounds were removed from ZMBHHP by ultrasonic extraction with 70% ethanol, and then analyzed by fast high-performance liquid chromatography (HPLC) coupled with time-of-flight mass spectrometry (TOFMS). The accurate-mass capability of the TOF analyzer allowed reliable confirmation of the identity of the detected compounds, normally with mass errors below 3 ppm in routine analysis. This mass accuracy was sufficient to verify the elemental compositions of the chemical constituents in ZMBHHP. With dynamic adjustment of fragmentor voltage in TOFMS, an efficient ion transmission was achieved to obtain the best sensitivity and abundant fragmentation. By accurate mass measurements for each molecular ion and subsequent fragment ions, a reliable identification and differentiation of 24 saponins, 3 xanthones, 1 anthraquinone and 2 alkaloids was described here, including four groups of isomers. It is concluded that this fast and sensitive HPLC/ESI-TOFMS technique is powerful in qualitative analysis of complex herbal medicines in terms of sensitivity, selectivity, resolving power, time savings and lower solvent consumption. Furthermore, the data gathered in this study may be helpful for understanding the synergistic nature of this herb pair in traditional Chinese medicine (TCM) and further pharmacokinetic studies of ZMBHHP.

Hydrolytic bacteria in mesophilic and thermophilic degradation of plant biomass

AbstractAdding plant biomass to a biogas reactor, hydrolysis is the first reaction step in the chain of biological events towards methane production. Maize silage was used to enrich efficient hydrolytic bacterial consortia from natural environments under conditions imitating those in a biogas plant. At 55–60°C a more efficient hydrolyzing culture could be isolated than at 37°C. The composition of the optimal thermophilic bacterial consortium was revealed by sequencing clones from a 16S rRNA gene library. A modified PCR‐RFLP pre‐screening method was used to group the clones. Pure anaerobic cultures were isolated. 70% of the isolates were related to Clostridium thermocellum. A new culture‐independent method for identification of cellulolytic enzymes was developed using the isolation of cellulose‐binding proteins. MALDI‐TOF/TOF analysis and end‐sequencing of peptides from prominent protein bands revealed cellulases from the cellulosome of C. thermocellum and from a major cellulase of Clostridium stercorarium. A combined culture of C. thermocellum and C. stercorarium was shown to excellently degrade maize silage. A spore preparation method suitable for inoculation of maize silage and optimal hydrolysis was developed for the thermophilic bacterial consortium. This method allows for concentration and long‐term storage of the mixed culture for instance for inoculation of biogas fermenters.

Comparison of MALDI TOF with conventional identification of clinically relevant bacteria

A completely new approach to diagnose microbial agents at least one day earlier based on mass spectrometric analysis becomes possible in the microbiology laboratory: MALDI TOF: matrix-assisted laser desorption/ionisation time-of-flight. Comparison between results of the new procedure with those obtained by conventional testing is mandatory. 204 clinical isolates grown on agar plates were analysed both, by the MALDI TOF Bruker microflex apparatus and by conventional identification using the VITEK II and API systems, both from bioMérieux. Of the identified isolates, 72 were gram-positive and 130 gram-negative; 2 were yeasts (candida). Concordance was seen with 61/72 (85%) of the Gram-positive bacteria and with 115/130 (88%) of the Gram-negative bacteria. In 27 samples (13.2%), a discrepancy of the species and/or genus was obvious. The discrepancy appeared with 16 gram-negative (12.2%) and with 11 gram-positive germs (15.3%, n.s.). In the latter group, 6 samples showed discordance with Streptococcus pneumoniae (MALDI) and Streptococcus mitis/oralis (conventional identification) constellation. Among gram-negative samples, most differences occurred on the species level only, e.g. Enterobacter cloacae versus Enterobacter kobei. In 5 cases, discordance was major and appeared on the genus level: Enterobacter/Raoultella, Streptococcus/Gemella, Pseumdomonas/Burkholderia, Microbacter/Sphingomonas and Candida/Cryptococcus. The most outstanding difference was Microbacterium arborescens (MALDI TOF) and Sphingomonas paucimobilis (conventional). Molecular biological identification of two Streptococcus mitis group bacteria confirmed the erroneous diagnosis by MALDI TOF of Streptococcus pneumoniae. Good comparability between MALDI TOF analysis and conventional identification procedures (86.8%) but special caution is needed when identifying streptococcal species.

Assessment of capillary electrophoresis TOF MS for a confident identification of peptides

CE on-line coupled to orthogonal accelerated TOF-MS (CE-oa-TOF-MS) is an emerging technique offering efficient charge-to-mass-based separations, as well as accurate and high-resolution mass measurements. Here, we investigated the main factors influencing the analysis of low molecular mass peptides using a sheath-flow electrospray ionization interface and several neuropeptides as model compounds. Moderate fragmentor voltage values of the oa-TOF-MS were crucial to maximize the production of molecular ions for optimum sensitivity precluding molecular fragmentation. However, the major fragments provided specific information that may result valuable for confirmatory purposes. Advantages and disadvantages of adding internal mass references to the sheath liquid for continuous mass-to-charge recalibration during the electrophoretic separations were discussed with regard to mass accuracy and sensitivity. Furthermore, several instrumental modes related to mass resolution were also examined. Finally, the method was validated for quantitative analysis of the studied neuropeptides in terms of repeatability, linearity and LODs. The results obtained using CE-oa-TOF-MS were compared with those obtained using CE coupled to other mass spectrometers. In addition to the simplicity and reliability on the identification, CE-oa-TOF-MS allowed improved repeatability and an around 10-fold improvement in sensitivity with respect to conventional reflectron TOF and ion trap mass analyzers.

Hydrolytically Stable Bioactive Synthetic Glycopeptide Homo- and Copolymers by Combination of NCA Polymerization and Click Reaction

The synthesis of poly(dl-propargylglycine) and poly(γ-benzyl-l-glutamate-co-dl-propargylglycine) was performed by NCA polymerization at 0 °C to yield well-defined polypeptides with polydispersity indices below 1.3. FTIR results confirm β-sheet and α-helical conformation of the homopolymer and copolymer, respectively. The subsequent glycosylation was achieved by Huisgen [3 + 2] cylcoaddition (“click” reaction) with azide-functional galactose. FTIR, NMR, SEC, and MALDI−ToF analyses verify the successful glycosylation and suggest a high efficiency of the click reaction. The homoglycopeptide was found to be water-soluble and to form aggregates in water above a critical concentration of 0.079 mg/mL. Selective lectin binding experiments confirmed that the glycopeptides can be used in biorecognotion applications. Moreover, the selective hydrolysis of the benzyl ester groups in the copolymer was achieved without loss of the galactose.

Redox-Active Ferrocenylboronium Polyelectrolytes with Main Chain Charge-Transfer Structure

A cationic poly(ferrocenylboronium) derivative was prepared by postpolymerization modification reaction of poly(ferrocenylbromoborane) (P1) with 4,4′-dinonyl-2,2′-bipyridyl (Rbipy). The polymer was found to show good stability under ambient conditions and was well soluble in polar organic solvents. Dialysis in MeOH allowed for isolation of a high molecular weight fraction that was further examined. The number-average of repeat units was estimated by GPC analysis relative to PS standards to be n =17, and MALDI−TOF analysis confirmed the chemical structure of the polymer repeat units. A characteristic purple color was observed in solution, which is due to charge-transfer from the electron-rich ferrocenyl to the electron-deficient bipyridylboronium moieties along the polymer main chain.

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.

Analysis of hemoglobin‐based oxygen carriers by CE‐UV/Vis and CE‐ESI‐TOF/MS

Blood doping involves the use of products that enhance the uptake, transport, or delivery of oxygen to the blood. One approach uses artificial oxygen carriers, known as hemoglobin-based oxygen carriers (HBOCs). This study describes an analytical strategy based on CE for detecting intact HBOCs in plasma samples collected for doping control. On-capillary detection was performed by UV/Vis at 415 nm, which offered detection selectivity for hemoproteins (such as hemoglobin and HBOCs). On-line ESI-MS detection with a TOF analyzer was further used to provide accurate masses on CE peaks and to confirm the presence of HBOCs. An immunodepletion sample preparation step was mandatory prior to analysis, in order to remove most abundant proteins that interfered with CE separation and altered the ESI process. This analytical method was successfully applied to plasma samples enriched with Oxyglobin, a commercially available HBOC used for veterinary purposes. Detection limits of 0.20 and 0.45 g/dL were achieved in plasma for CE-UV/Vis at 415 nm and CE-ESI-TOF/MS, respectively.