Software-Assisted Data Processing Workflow for Intact Glycoprotein Mass Spectrometry

Intact protein analysis by mass spectrometry is important for several applications such as assessing post-translational modifications and biotransformation. In particular, intact protein analysis allows the detection of proteoforms that are commonly missed by other approaches such as proteolytic digestion followed by bottom-up analysis. Two quantification methods are mainly used for intact protein data quantification, namely the extracted ion and deconvolution approaches. However, a consensus with regard to a single best practice for intact protein data processing is lacking. Furthermore, many data processing tools are not fit-for-purpose and, as a result, the analysis of intact proteins is laborious and lacks the throughput required to be implemented for the analysis of clinical cohorts. Therefore, in this study, we investigated the application of a software-assisted data analysis and processing workflow in order to streamline intact protein integration, annotation, and quantification via deconvolution. In addition, the assessment of orthogonal data sets generated via middle-up and bottom-up analysis enabled the cross-validation of cleavage proteoform assignments present in seminal prostate-specific antigen (PSA). Furthermore, deconvolution quantification of PSA from patients' urine revealed results that were comparable with manually performed quantification based on extracted ion electropherograms. Overall, the presented workflow allows fast and efficient processing of intact protein data. The raw data is available on MassIVE using the identifier MSV000086699.</p

Formosolv pretreatment to fractionate Paulownia wood following a biorefinery approach: isolation and characterization of the lignin fraction

Paulownia is a rapid-growth tree with a high biomass production rate per year and low demand of water, which make it very suitable for intercropping systems, as it protects the crops from adverse climatic conditions, benefiting the harvest yields. Moreover, these characteristics make Paulownia a suitable raw material able to be fractionated in an integrated biorefinery scheme to obtain multiple products using a cascade conversion approach. Different delignification pretreatments of biomass have been purposed as a first stage of a lignocellulosic biorefinery. In this study, the formosolv delignification of Paulownia wood was investigated using a second order face-centered factorial design to assess the effects of the independent variables (concentrations of formic and hydrochloric acids and reaction time) on the fractionation of Paulownia wood. The maximum delignification achieved in this study (78.5%) was obtained under following conditions: 60 min, and 95% and 0.05% formic and hydrochloric acid, respectively. In addition, the remained solid phases were analyzed to determine their cellulose content and cooking liquors were also chemically analyzed and characterized. Finally, the recovered lignin by precipitation from formosolv liquor and the pristine lignin (milled wood lignin) in Paulownia wood were characterized and compared by the following techniques FTIR, NMR, high-performance size-exclusion chromatography (HPSEC) and TGA. This complete characterization allowed verifying the capacity of the formosolv process to act on the lignin, causing changes in its structure, which included both phenomena of depolymerization and condensation.This research was funded by MINECO (Spain) in the framework of the projects “Development of processes for the integral use of fast-growing biomass by means of the production of bioethanol and chemical products” with reference CTQ2012-30855 and “Multistage processes for the integral benefit of macroalgal and vegetal biomass” with reference CTM2015-68503-R, by Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) through the contract ED431C 2017/62-GRC to Competitive Reference Group BV1, and by the CITACA Strategic Partnership ED431E 2018/07, programs partially funded by European Regional Development Fund (FEDER).info:eu-repo/semantics/publishedVersio

Linking the concentrations of itraconazole and 2-hydroxypropyl-β-cyclodextrin in human intestinal fluids after oral intake of Sporanox^®

In a previously performed small-scale clinical study, healthy volunteers were asked to ingest an oral solution of itraconazole (Sporanox®) containing 40% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) (i) with or (ii) without a standardized volume of water (240 mL) after which gastrointestinal and blood samples were collected. Although omitting water during the administration of Sporanox® resulted in noticeably higher duodenal concentrations of itraconazole, systemic exposure was almost unaffected. It is assumed that this discrepancy can be explained by differences in the extent of entrapment of itraconazole in the duodenum caused by differential complexation depending on the concentration of cyclodextrins. To further substantiate this hypothesis, the quantification of HP-β-CD concentrations in the aspirated intestinal fluids was performed by LC-MS/MS. When comparing the intestinal concentrations of itraconazole and HP-β-CD for one single healthy volunteer (HV02) in both test conditions, an excellent correlation was observed (Spearman's rank coefficient of 0.96). Moreover, the data suggest that, similar to aqueous buffer media, also in human intestinal fluids a non-linear relationship exists between itraconazole solubility and HP-β-CD concentration (Ap-type profile; Spearman's rank coefficient of 0.78), indicating that higher order complexes are formed at higher concentrations of HP-β-CD. This difference in extent of entrapment in the inclusion complexes helps to understand the observed impact of water intake on precipitation and permeation behavior of itraconazole in man. Without water intake, higher HP-β-CD concentrations resulted in less precipitation and increased duodenal concentrations of itraconazole. On the other hand, the stronger interaction at higher HP-β-CD concentrations reduced the free fraction of the drug explaining that increased intraluminal concentrations of itraconazole were not translated into an enhanced uptake. In conclusion, quantifying the concentrations of the solubilizing agent HP-β-CD in human intestinal fluids appeared to be of crucial importance to interpret the intraluminal behavior of an orally administered cyclodextrin-based solution

Development of a surface plasmon resonance sensor for coupling to capillary electrophoresis allowing affinity assessment of protein mixture components

Surface plasmon resonance (SPR) currently is the major platform to study protein–protein interactions, but it lacks the selectivity to distinguish between binding components within one sample. Capillary electrophoresis (CE) can provide efficient separation of intact proteins under near-physiological conditions. We have hyphenated CE with SPR to achieve affinity assessment of mixture components. A microfluidic flow cell allowing straightforward coupling of CE and SPR was developed. Initial testing with non-interacting dyes showed good performance using a flow-cell channel volume of 100 nL until the detection point. Appropriate closing of the CE electric circuit was achieved using the SPR gold-sensor as grounding electrode. Division of the (bio)sensor into an electrode part (providing grounding) and a detection part (bearing the affinity surface) was crucial to avoid disturbance of the SPR signal by the CE voltage. This approach permitted CE separation and binding assessment for separation voltages up to 30 kV. Human serum albumin (HSA) or aprotinin were immobilized on carboxymethyldextran hydrogel-coated gold sensors and target proteins (anti-HSA, and trypsin and α-chymotrypsin, respectively) were analyzed. Efficient CE separation of the intact protein analytes was accomplished under native conditions by employing neutral and positively-charged capillary coatings. Selective binding of separated proteins to the target surface could be monitored by SPR down to 2 ng of injected protein. Regeneration of the biosensor surface was achieved by an on-line rising, allowing repeatable CE-SPR analyses of proteins with RSDs below 1% and 5% for migration time and signal intensity, respectively

Enantiomeric separation of ornithine in complex mixtures of amino acids by EKC with off-line derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

A new analytical methodology was developed by EKC enabling the fast enantiomeric separation of\ud Ornithine in complex mixtures of amino acids. A previous derivatization step with 6-aminoquinolyl-Nhydroxysuccinimidyl\ud carbamate (AQC) was achieved to enable the sensitive UV detection of amino acids\ud as well as to make possible their interaction with the CDs employed as chiral selectors. A dual CD system\ud containing an anionic and a neutral CD in phosphate buffer at acid pH showed a high resolving power\ud allowing the enantiomeric separation of 18 protein amino acids and Orn. The method was applied to the\ud analysis of fermented foods to investigate the extent of the presence of Orn enantiomers.Authors thank the Ministry of Education of Science (Spain) and\ud the Comunidad Autonoma de Madrid (Spain) for research projects CTQ2006-03849/BQU and S-0505/AGR-0312, respectively. Carmen Garcıa-Ruiz thanks the Ministry of Science and Technology (Spain) for the Ramon y Cajal program (RYC-2003-001). Ana Belen Martínez \ud Giron and Elena Domínguez-Vega thank the University of Alcala for their pre-doctoral grants

high resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography mass spectrometry

Abstract Glycosylation is considered a critical quality attribute of therapeutic proteins. Protein heterogeneity introduced by glycosylation includes differences in the nature, number and position of the glycans. Whereas analysis of released glycans and glycopeptides provides information about the composition and/or position of the glycan, intact glycoprotein analysis allows assignment of individual proteoforms and co-occurring modifications. Yet, resolving protein glycoforms at the intact level is challenging. We have explored the capacity of hydrophilic liquid chromatography-mass spectrometry (HILIC-MS) for assessing glycosylation patterns of intact pharmaceutical proteins by analyzing the complex glycoproteins interferon-beta-1a (rhIFN-β − 1a) and recombinant human erythropoietin (rhEPO). Efficient glycoform separation was achieved using a superficially-porous amide HILIC stationary phase and trifluoroacetic acid (TFA) as eluent additive. In-source collision-induced dissociation proved to be very useful to minimize protein-signal suppression effects by TFA. Direct injection of therapeutic proteins in aqueous formulation was possible without causing extra band dispersion, provided that the sample injection volume was not larger than 2 μL. HILIC-MS of rhIFN-β − 1a and rhEPO allowed the assignment of, respectively, 15 and 51 glycoform compositions, next to a variety of posttranslational modifications, such as succinimide, oxidation and N-terminal methionine-loss products. MS-based assignments showed that neutral glycan units significantly contributed to glycoform separation, whereas terminal sialic acids only had a marginal effect on HILIC retention. Comparisons of HILIC-MS with the selectivity provided by capillary electrophoresis-MS for the same glycoproteins, revealed a remarkable complementarity of the techniques. Finally it was demonstrated that by replacing TFA for difluoroacetic acid, peak resolution somewhat decreased, but rhEPO glycoforms with relative abundances below 1% could be detected by HILIC-MS, increasing the overall rhEPO glycoform coverage to 72

Affinity capillary electrophoresis - mass spectrometry permits direct binding assessment of IgG and FcγRIIa in a glycoform-resolved manner

The impact of antibody glycoforms on FcγRIIa activation and immune responses is poorly understood. Yet, glycoform binding assessment remains one of the major analytical challenges requiring long enrichment or glycoengineering steps. Here, we developed and applied an affinity capillary electrophoresis-mass spectrometry approach to selectively assess the binding of different antibody glycoforms to the FcγIIa receptor without the need of glycoengineering. The approach required only low microgram amounts of antibody and receptor and enables assessing the binding of high and low-abundance glycoforms. The approach indicated clear differences in binging between doubly-, hemi-glycosylated and non-glycosylated antibodies as well as for mutated (Leu234Ala, Leu235Ala - Pro329-Gly (LALA-PG)) IgG1 antibodies silenced for Fcγ binding. The LALA-PG mutated antibody showed no binding to the FcγIIa receptor (excluding potential non-specific binding effects) while the non-glycosylated IgG1 showed a strongly reduced, but still minor binding. The highest binding affinity was for the antibody carrying two complex-type glycans. Man5 glycans resulted in decreased binding compared to complex-type glycans, with the lowest binding for the IgG containing two Man5. For complex-type glycans, galactosylation showed a subtle increase in binding to the FcγIIa receptor, and sialylation showed an increase in binding for lower sialylated species. Fucosylation did not influence binding to the FcγIIa receptor. Finally, the assay was evaluated for the two variants of the FcγRIIa receptor (allotypes H131 and R131) showing highly comparable glycoform selectivity. Overall, the proposed approach allows the direct comparison of binding affinities of different antibody species in mixtures promising a fast establishment of their structure-function relationships

MKK6 controls T3-mediated browning of white adipose tissue

El aumento de la capacidad termogénica del tejido adiposo para mejorar el gasto de energía del organismo se considera una estrategia terapéutica prometedora para combatir la obesidad. Aquí nosotros informe que la expresión del activador MAPK p38 MKK6 está elevada en el tejido adiposo blanco de individuos obesos. Usando animales knockout y shRNA, mostramos que la eliminación de Mkk6 aumenta el gasto de energía y la capacidad termogénica del tejido adiposo blanco, protegiendo a los ratones contra la obesidad inducida por la dieta y el desarrollo de la diabetes. La eliminación de Mkk6 aumenta la expresión de UCP1 estimulada por T3 en los adipocitos, lo que aumenta su capacidad termogénica. De manera mecánica, demostramos que, en el tejido adiposo blanco, p38 se activa mediante una ruta alternativa que involucra AMPK, TAK y TAB. Nuestros resultados identifican MKK6 en los adipocitos como un posible objetivo terapéutico para reducir la obesidad.Increasing the thermogenic capacity of adipose tissue to enhance organismal energy expenditure is considered a promising therapeutic strategy to combat obesity. Here, we report that expression of the p38 MAPK activator MKK6 is elevated in white adipose tissue of obese individuals. Using knockout animals and shRNA, we show that Mkk6 deletion increases energy expenditure and thermogenic capacity of white adipose tissue, protecting mice against diet-induced obesity and the development of diabetes. Deletion of Mkk6 increases T3-stimulated UCP1 expression in adipocytes, thereby increasing their thermogenic capacity. Mechanistically, we demonstrate that, in white adipose tissue, p38 is activated by an alternative pathway involving AMPK, TAK, and TAB. Our results identify MKK6 in adipocytes as a potential therapeutic target to reduce obesity.• Guadalupe Sabio Buzo y Rebeca Acin Pérez pertenecen a Programa Ramón y Cajal • Elisa Manieri pertenece a Caixa • Ministerio de Economía y Competitividad. Proyecto FPI BES-2014-069332, para Valle Montalvo Romeral • Ministerio de Economía y Competitividad. Proyecto FPI BES-2011-043428, para Edgar Bernardo • Ministerio de Economía y Competitividad y FEDER SAF2016-79126-R y Comunidad de Madrid S2010 / BMD-2326, para Guadalupe Sabio Buzo • ISCIII y FEDER, PI10 / 01692 e I3SNS-INT12 / 049, para Miguel Marcos Martín • Junta de Castilla y León GRS 681 / A / 11, para Lourdes Hernández Cosido • Ministerio de Economía y Competitividad. BFU2015-70664-R, Xunta de Galicia 2015-CP080 y PIE13 / 00024, y ERC281408, para Rubén Nogueiras Pozo • Unión Europea. Becas europeas UE0 / MCA1108 y UE0 / MCA1201; y la Comunidad de Madrid CAM / API1009, para Rubén Nogueiras Pozo • Junta de Extremadura y FEDER BR15164, para Francisco Centeno Velázquez • Ministerio de Economía y Competitividad. . BFU2013-46109-R, para Clara V. Álvarez Villamarín • European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. ERC 260464peerReviewe

Phylogeny and phylogeography of a recent HIV-1 subtype F outbreak among men who have sex with men in Spain deriving from a cluster with a wide geographic circulation in Western Europe

This work received support from the Dirección General de Farmacia, Ministerio de Sanidad, Servicios Sociales e Igualdad, Government of Spain, grant EC11-272; European Network of Excellence EUROPRISE (Rational Design of HIV Vaccines and Microbicides), grant LSHP-CT-2006-037611; European Research Infrastructures for Poverty Related Diseases (EURIPRED). Seventh Framework Programme: FP7-Capacities-infrastructures-2012-1, grant agreement 312661; Instituto de Salud Carlos III, Subdirección General de Evaluación, and Fondo Europeo de Desarrollo Regional (FEDER), Plan Nacional I + D + I, through project RD12/0017/0026; Consellería de Sanidade, Government of Galicia, Spain (MVI 1291/08); and the Osakidetza-Servicio Vasco de Salud, Basque Country, Spain (MVI-1255-08). Marcos Pérez-Losada was supported by a DC D-CFAR Research Award from the District of Columbia Developmental Center for AIDS Research (P30AI087714) and by an University Facilitating Fund award from George Washington University. Aurora Fernández-García is supported by CIBER in Epidemiology and Public Health, Instituto de Salud Carlos III, Madrid, Spain.We recently reported the rapid expansion of an HIV-1 subtype F cluster among men who have sex with men (MSM) in the region of Galicia, Northwest Spain. Here we update this outbreak, analyze near full-length genomes, determine phylogenetic relationships, and estimate its origin. For this study, we used sequences of HIV-1 protease-reverse transcriptase and env V3 region, and for 17 samples, near full-length genome sequences were obtained. Phylogenetic analyses were performed via maximum likelihood. Locations and times of most recent common ancestors were estimated using Bayesian inference. Among samples analyzed by us, 100 HIV-1 F1 subsubtype infections of monophyletic origin were diagnosed in Spain, including 88 in Galicia and 12 in four other regions. Most viruses (n = 90) grouped in a subcluster (Galician subcluster), while 7 from Valladolid (Central Spain) grouped in another subcluster. At least 94 individuals were sexually-infected males and at least 71 were MSM. Seventeen near full-length genomes were uniformly of F1 subsubtype. Through similarity searches and phylogenetic analyses, we identified 18 viruses from four other Western European countries [Switzerland (n = 8), Belgium (n = 5), France (n = 3), and United Kingdom (n = 2)] and one from Brazil, from samples collected in 2005?2011, which branched within the subtype F cluster, outside of both Spanish subclusters, most of them corresponding to recently infected individuals. The most probable geographic origin and age of the Galician subcluster was Ferrol, Northwest Galicia, around 2007, while the Western European cluster probably emerged in Switzerland around 2002. In conclusion, a recently expanded HIV-1 subtype F cluster, the largest non-subtype B cluster reported in Western Europe, continues to spread among MSM in Spain; this cluster is part of a larger cluster with a wide geographic circulation in diverse Western European countries.Publisher PDFPeer reviewe

Autoevalaución y coevaluación del proceso del trabajo en equipo en el marco del EEES

Memoria ID-0134. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2015-2016