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The bacterial cell surface (surfaceome) is the first site encountered by immune cells and is thus an important site for immune recognition. As such, the characterization of bacterial surface proteins can lead to the discovery of novel antigens for potential vaccine development. In this chapter, we describe a rapid 5-min surface shaving proteomics protocol where live bacterial cells are incubated with trypsin and surface peptides are "shaved" off. The shaved peptides are subsequently identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several checkpoints, including colony forming unit (CFU) counts, flow cytometry, and a false positive unshaved control, are introduced to ensure cell viability/membrane integrity are maintained and that proteins identified are true surface proteins. The protein topology of shaved peptides can be bioinformatically confirmed for surface location. Surface shaving facilitates identification of surface proteins expressed under different conditions, by different strains as well as highly abundant essential and immunogenic bacterial surface antigens for potential vaccine development.

Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1—non-muscle invasive bladder cancer (NMIBC), and T2/T3—muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers., Open access funding provided by Uppsala University., This work was supported by Magnus Bergvall Foundation (S.B.L, 2017-02330, 2018-02726, 2019-03296), Clas Groschinsky memory foundation (S.B.L., M1603, M1742), and Swedish Foundation for Strategic research (S.B.L., SB16-0039). A.V. would like to acknowledge the Ministry of Science and Innovation for a “Juan de la Cierva” postdoctoral Grant.

13 páginas, 5 figuras, 2 tablas, [Background] The argasid tick Ornithodoros moubata is the main vector in mainland Africa of African swine fever virus and the spirochete Borrelia duttoni, which causes human relapsing fever. The elimination of populations of O. moubata would contribute to the prevention and control of these two serious diseases. Anti-tick vaccines are an eco-friendly and sustainable means of eliminating tick populations. Tick saliva forms part of the tick-host interface, and knowledge of its composition is key to the identification and selection of vaccine candidate antigens. The aim of the present work is to increase the body of data on the composition of the saliva proteome of adult O. moubata ticks, particularly of females, since in-depth knowledge of the O. moubata sialome will allow the identification and selection of novel salivary antigens as targets for tick vaccines., [Methods] We analysed samples of female and male saliva using two different mass spectrometry (MS) approaches: data-dependent acquisition liquid chromatography-tandem MS (LC–MS/MS) and sequential window acquisition of all theoretical fragment ion spectra–MS (SWATH-MS). To maximise the number of proteins identified, a proteomics informed by transcriptomics analysis was applied using the O. moubata salivary transcriptomic dataset previously obtained by RNA-Seq., [Results] SWATH-MS proved to be superior to LC–MS/MS for the study of female saliva, since it identified 61.2% more proteins than the latter, the reproducibility of results was enhanced with its use, and it provided a quantitative picture of salivary components. In total, we identified 299 non-redundant proteins in the saliva of O. moubata, and quantified the expression of 165 of these in both male and female saliva, among which 13 were significantly overexpressed in females and 40 in males. These results indicate important quantitative differences in the saliva proteome between the sexes., [Conclusions] This work expands our knowledge of the O. moubata sialome, particularly that of females, by increasing the number of identified novel salivary proteins, which have different functions at the tick–host feeding interface. This new knowledge taken together with information on the O. moubata sialotranscriptome will allow a more rational selection of salivary candidates as antigen targets for tick vaccine development., This research was funded by project RTI2018-098297-B-I00, granted by the Spanish Ministry of Science, Innovation and Universities and Project CLU-2019-05-IRNASA/CSIC Unit of Excellence, funded by the Junta de Castilla y León and co-financed by the European Union (Europe drives our growth; European Regional Development Fund).

We report on a potential method to separate sugars by using the specific interaction between fullerenes and saccharides in liquid chromatography (LC). Aromatic rings with high electron density are believed to interact strongly with saccharides due to CH–π and/or OH–π interactions. In this study, the fullerene-bonded columns were used to separate saccharides by LC under aqueous conditions. As a result, 2-aminobenzamide-labeled glucose homopolymer (Glcs) was effectively separated by both C60 and C70 columns in the range of Glc-1 to Glc-20 and high blood glucose level being retained in greater quantity. Furthermore, similar separations were identified by LC–mass spectrometry with non-labeled glucose homopolymers. Theoretical study based on molecular dynamics and DFT calculation demonstrated that a supramolecular complex of saccharide–fullerene was formed through CH–π and/or OH–π interactions, and that the interactions between saccharide and fullerene increase with the increase units of the saccharide. Additionally, the C60 column retained disaccharides containing maltose, trehalose, and sucrose. In this case, it was assumed that the retention rates were determined by the difference of the dipole moment in each saccharide. These results suggest that the dipole-induced dipole interaction was dominant, and that maltose—with the higher dipole moment—was more strongly retained compared to other disaccharides having lower dipole moment., フラーレンを用いた糖鎖の分離手法を開発 --抗体等の糖タンパク質分離への応用に期待--. 京都大学プレスリリース. 2020-08-26.

The establishment of a reference method for the determination of the allergen protein content in a processed food material has been explored. An analytical approach was developed to enable the comparability of food allergen measurement results expressed in a decision-relevant manner. A proof of concept is here presented, resulting in quantity values for the common measurand, namely ‘mass of total allergen protein per mass of food’. The quantities are determined with SI traceability to enable the comparability of reported results. A method for the quantification of total milk protein content in an incurred baked food at a concentration level clinically relevant is presented. The strategy on how to obtain the final analytical result is outlined. Challenges associated with this method are discussed, in particular the optimal extraction of the marker proteins, the complete digestion and release of the peptides in an equimolar fashion, the use of conversion factors to translate the amount of measured proteins into total milk protein and the estimation of the uncertainty contributions as well as of the combined uncertainty of the final result. The implementation of such a reference method for the determination of the total allergen content in a processed food is an important step, which will provide comparable measurement data of relevance to risk assessors. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02959-0) contains supplementary material, which is available to authorized users.

Background Toxocara canis, a globally distributed roundworm, can cause debilitating disease in dogs and humans; however, little is known about the metabolomic response of the hosts to T. canis infection. There is an increasing need to understand the metabolic mechanisms underlying the pathogenesis of T. canis infection in dogs. Here, we examined the metabolomic changes in Beagle dogsʼ serum following T. canis infection using LC-MS/MS. Results The metabolic profiles of Beagle dogsʼ serum were determined at 12 h, 24 h, 10 d and 36 d after oral infection with 300 infectious T. canis eggs by LC-MS/MS. We tested whether the T. canis-associated differentially abundant metabolites could distinguish the serum of infected dogs from controls, as measured by the area under the receiver operating characteristic (ROC) curve (AUC). The differentially expressed metabolites were further evaluated by principal components analysis and pathway enrichment analysis. A total of 5756 and 5299 ions were detected in ESI+ and ESI− mode, respectively. ROC curve analysis revealed nine and five metabolite markers, at 12 hpi and 24 hpi to 36 dpi, respectively, with potential diagnostic value for toxocariasis. The levels of taurocholate, estradiol, prostaglandins and leukotriene were significantly changed. Primary bile acid biosynthesis pathway, steroid hormone biosynthesis pathway and biosynthesis of unsaturated fatty acids pathway were significantly altered by T. canis infection. Conclusions These findings show that T. canis infection can induce several changes in the dog serum metabolome and that the metabolic signature associated with T. canis infection in dogs has potential for toxocariasis diagnosis.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (cyclodextrans) from starch even in the absence of dextran. Cycloisomaltooligosaccharide glucanotransferase synthesizes cycloisomaltooligosaccharides exclusively from an α-(1 → 6)-consecutive glucose chain consisting of at least four molecules. Starch is not a substrate of this enzyme. Therefore, we predicted that the bacterium possesses another enzyme system for extending α-(1 → 6)-linked glucoses from starch, which can be used as the substrate for cycloisomaltooligosaccharide glucanotransferase, and identified the transglucosylation enzyme Ps6GT31A. We purified Ps6GT31A from the bacterial culture supernatant, cloned its corresponding gene, and characterized the recombinant enzyme. Ps6GT31A belongs to glycoside hydrolase family 31, and it liberates glucose from the non-reducing end of the substrate in the following order of activity: α-(1 → 4)-> α-(1 → 2)- > α-(1 → 3)- > α-(1 → 6)-glucobiose and maltopentaose > maltotetraose > maltotriose > maltose. Ps6GT31A catalyzes both hydrolysis and transglucosylation. The resulting transglucosylation compounds were analyzed by high-performance liquid chromatography and mass spectrometry. Analysis of the initial products by 13C nuclear magnetic resonance spectroscopy revealed that Ps6GT31A had a strong α-(1 → 4) to α-(1 → 6) transglucosylation activity. Ps6GT31A elongated α-(1 → 6)-linked glucooligosaccharide to at least a degree of polymerization of 10 through a successive transglucosylation reaction. Eventually, cycloisomaltooligosaccharide glucanotransferase creates cycloisomaltooligosaccharides using the transglucosylation products generated by Ps6GT31A as the substrates. Our data suggest that Ps6GT31A is the key enzyme to synthesize α-(1 → 6)-glucan for cycloisomaltooligosaccharide production in dextran-free environments.

The developed method was evaluated for the determination of 10 antibiotics belonging to four chemical classes (fluoroquinolones, sulfonamides, lincosamides, and metoxybenzylpyrimidines) and six of their metabolites in four vegetable matrices (lettuce, tomato, cauliflower, and broad beans). The reported method detection limits were sufficiently low (0.1–5.8 ng/g dry weight) to detect target compounds in vegetables under real agricultural practices. Absolute and relative recovery values ranged from 40 to 118% and from 70 to 118%, respectively, for all targeted compounds at the spike level of 100 ng/g dry weight. Regarding method precision, the highest relative standard deviation (RSD) was obtained for enrofloxacin in lettuce (20%), while for the rest of the compounds in all matrices, the RSD values were below 20% for the same spike level. Matrix effects, due to electrospray ionization, ranged from − 26 to 29% for 85% of all estimated values. In a field study, four of the 10 targeted antibiotics were detected in tested vegetables. For the first time, antibiotic metabolites were quantified in vegetables grown under real field conditions. More specifically, decarboxyl ofloxacin and TMP304 were detected in tomato fruits (1.5 ng/g dry weight) and lettuce leaves (21.0–23.1 ng/g dry weight), respectively. It is important to remark that the concentration of TMP304 was five times higher than that from the parental compound, emphasizing the importance of metabolite analysis in monitoring studies. Therefore, the method provided a robust, reliable, and simple-to-use tool that could prove useful for routine multiclass analysis of antibiotics and their metabolites in vegetable samples. [Figure not available: see fulltext.]. © 2019, Springer-Verlag GmbH Germany, part of Springer Nature., The work presented in this paper is part of a project that has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 675530. The authors also gratefully acknowledge the financial support of the Spanish Ministry of Economy, Industry, and Competitiveness (MEIC) through Project AGL2014-59353-R. Funding This study was funded by H2020 MSCA grant agreement 675530 and the MEIC project nr. AGL2014-59353-R.

The green microalga Haematococcus pluvialis has been widely studied due to its capacity to accumulate great amounts of astaxanthin, a high-value carotenoid with biological activities. In the present work, two green compressed fluid–based processes, pressurized liquid extraction (PLE) and supercritical antisolvent fractionation (SAF), are integrated to obtain an astaxanthin-enriched extract from this microalga. PLE was carried out using pressurized ethanol as solvent, for 20 min, at 10 MPa, and 50 °C as extraction temperature. Subsequently, the obtained extract was processed by SAF to further purify the carotenoid fraction. The SAF process was optimized using a 3-level factorial experimental design and considering three experimental variables: (i) CO2 pressure (10–30 MPa), (ii) percentage of water in the PLE extract (20–50%), and (iii) PLE extract/supercritical-CO2 flow rate ratio (0.0125–0.05). Total carotenoid content was evaluated in both extracts and raffinates. Best results were obtained at 30 MPa, 0.05 feed/SC-CO2 mass flow rate, and 20% (v/v) of water in the feed solution, achieving values of 120.3 mg g−1 carotenoids in extract (in the SAF extract fraction), which were significantly higher than those obtained in the original PLE extract. In parallel, a new fast two-dimensional comprehensive liquid chromatography (LC×LC) method was optimized to get the full carotenoid profile of these extracts in less than 25 min. This is the first time that the use of a C30 column is reported in an on-line LC×LC system., The authors would like to thank projects ABACUS (Algae for a Biomass Applied to the production of added value compounds—funded by the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 745668) and AGL2017-89417-R (MINECO, Spain) for funding.

© The Author(s) 2019., Some molecular chaperones are involved not only in assisting the folding of proteins but also, given appropriate conditions, in their degradation. This is the case for Hsp70 and Hsp90 which, in concert with the cochaperone CHIP, direct their bound substrate to degradation through ubiquitination. We generated complexes between the chaperones (Hsp70 or Hsp90), the cochaperone CHIP and, as substrate, a p53 variant containing the GST protein (p53-TMGST). Both ternary complexes (Hsp70:p53-TMGST:CHIP and Hsp90:p53-TMGST:CHIP) ubiquitinated the substrate at a higher efficiency than in the absence of the chaperones. The 3D structures of the two complexes, obtained using a combination of cryoelectron microscopy and crosslinking mass spectrometry, showed the substrate located between the chaperone and the cochaperone, suggesting a ubiquitination mechanism in which the chaperone-bound substrate is presented to CHIP. These complexes are inherently flexible, which is important for the ubiquitination process., This research was supported by the grant BFU2016-75984 (AEI/FEDER, EU) to JMV. We thank Diamond Light Source for access to beamline EM16943 and EM15997 that contributed to the results presented here. The CRG/UPF Proteomics Unit is a member of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I + D + i 2013–2016, funded by ISCIII and ERDF. We acknowledge support from the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013–2017”, SEV-2012–0208, and “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2017SGR595).

Some clinical trials showed that omega-3 fatty acid (FA) reduced cardiovascular events, but it remains unknown whether omega-3 FA supplementation changes the composition of FAs and their metabolites in the heart and how the changes, if any, exert beneficial effects on cardiac structure and function. To clarify these issues, we supplied omega-3 FA to mice exposed to pressure overload, and examined cardiac structure and function by echocardiography and a proportion of FAs and their metabolites by gas chromatography and liquid chromatography-tandem mass spectrometry, respectively. Pressure overload induced cardiac hypertrophy and dysfunction, and reduced concentration of all FAs’ components and increased free form arachidonic acid and its metabolites, precursors of pro-inflammatory mediators in the heart. Omega-3 FA supplementation increased both total and free form of eicosapentaenoic acid, a precursor of pro-resolution mediators and reduced free form arachidonic acid in the heart. Omega-3 FA supplementation suppressed expressions of pro-inflammatory cytokines and the infiltration of inflammatory cells into the heart and ameliorated cardiac dysfunction and fibrosis. These results suggest that omega-3 FA-induced changes of FAs composition in the heart have beneficial effects on cardiac function via regulating inflammation.

Cobalamin deficiency represents a health issue for vegetarians, especially vegans, if supplements are not consumed. Vitamin B12 serum levels, traditionally used to assess the vitamin B12 status, can be normal under functional deficiency conditions. In this regard, methylmalonic acid (MMA) has proven to be a more specific marker to detect subclinical vitamin B12 deficiency. In this study, we present for the first time the cobalamin status of Spanish vegetarians using both vitamin B12 and MMA markers, and the effects of the plant-based diet and the intake of vitamin B12 supplements. Healthy adults were recruited (n = 103, 52% vegans). Dietary preferences and use of supplements were assessed by questionnaires and serum samples were collected and stored. Vitamin B12 was measured by chemiluminiscence and MMA by liquid chromatography tandem mass spectrometry (LC-MS/MS) using solid phase extraction for sample preparation. Obtained values, median (IQR), were: vitamin B12, 278.9 (160.2) pmol/l and MMA, 140.2 (78.9) nmol/l. No significant differences between lacto-ovo vegetarians and vegans were observed. Considering these two markers, 10% of the participants were mild vitamin B12 deficient. Supplementation (75% of the participants) was associated with higher vitamin B12 (p, The study was supported by a research project financed by Zamdeh Laboratories (Veggunn study). A. Gallego-Narbón was funded by the Youth Employment Initiative (YEI) from the European Social Fund (ESF).

Polymers are used in high amounts in a wide range of applications from biomedicine to industry. Because of the growing awareness of the increasing amounts of plastic wastes in the aquatic environment during recent years, the evaluation of their biodegradability deserves special attention. In the past, most efforts were dedicated to studying the biodegradation of polyesters in soil and compost, while very little research has been conducted on their fate in wastewater. Here, we assessed the ability of bacterial communities residing in the aerobic and denitrification tank from a wastewater treatment plant (WWTP) to degrade the polymeric ester polycaprolactone diol (PCLD; average molecular weight of 1250 Da). Following the incubation of the solid polymer in WWTP tanks, matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used to provide evidence for hydrolytic reactions and to study differences in the spatial degradation on the PCLD surface. It was demonstrated that regardless of the wastewater type, the chemical structure on the PCLD surface underwent modifications after 7 days of exposure. Apart from the parent PCLD peak series in MALDI-MSI mass spectra, the presence of a second oligomer series with mass peaks spaced by m/z 114 (as in PCLD) was observed. It was proposed to correspond to polycaprolactone (PCL) originating from the hydrolytic cleavage of the diethylene glycol from PCLD. Their ion masses were detected at m/z 104 below the PCLD peaks and their structures were proposed as PCL cyclized oligomers. Differences in the spatial distribution of low MW ions (, This study has been financially supported by the EU through the FP7 project GLOBAQUA (Grant Agreement No 603629), and by the Generalitat de Catalunya (Consolidated Research Groups “2014 SGR 418—Water and Soil Quality Unit” and 2014 SGR 291—ICRA). It reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein.

Endometriosis is a common, complex gynecologic disorder characterized by the presence of endometrial-like tissues at extrauterine sites. Elevation in protein and lipid mediators of inflammation including oxylipins and cytokines within the peritoneum characterize the inflamed pelvic region and may contribute to the survival and growth of displaced endometrial tissues. The presence of a clinically silent but molecularly detectable systemic inflammation in endometriosis has been proposed. Thus, we examined serum oxylipin and immunomodulatory protein levels in 103 women undergoing laparoscopy to evaluate systematically any involvement in systemic pathophysiological inflammation in endometriosis. Oxylipin levels were similar between women with and without endometriosis. Stratification by menstrual phase or severity did not offer any difference. Women with ovarian endometriosis had significantly lower 12-HETE relative to peritoneal endometriosis (−50.7%). Serum oxylipin levels were not associated with pre-operative pain symptoms. Changes to immunomodulatory proteins were minimal, with IL-12(p70), IL-13 and VEGF significantly lower in mild endometriotic women compared to non-endometriotic women (−39%, −54% and −76% respectively). Verification using C-reactive protein as a non-specific marker of inflammation further showed similar levels between groups. The implications of our work suggest pro-inflammatory mediators in the classes studied may have potentially limited value as circulating biomarkers for endometriosis, suggesting of potentially tenuous systemic inflammation in endometriosis., Singapore. National Medical Research Council (NMRC/BNIG/2033/2015), SingHealth Foundation (SHF/FG560P/2014)