scholarly journals Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches

2021 ◽  
Author(s):  
Yang Yang ◽  
Jake W. Pawlowski ◽  
Ian J. Carrick ◽  
Igor Kaltashov
Keyword(s):  
Disulfide Bonds ◽  
Heterogeneous Systems ◽  
Structural Diversity ◽  
Mass Range ◽  
Protein Characterization ◽  
Charge Reduction ◽  
Average Mass ◽  
Significant Difference ◽  
Intact Mass

Intact-mass measurements are becoming an increasingly popular in mass spectrometry (MS) based protein characterization, as they allow the entire complement of proteoforms to be evaluated within a relatively short time. However, applications of this approach are currently limited to systems exhibiting relatively modest degrees of structural diversity, as the high extent of heterogeneity frequently prevents straightforward MS measurements. Incorporation of limited charge reduction into electrospray ionization (ESI) MS measurements provides an elegant way to obtain meaningful information on most heterogeneous systems, yielding not only the average mass of the protein, but also the mass range populated by various proteoforms. Application of this approach to characterization of two different phenotypes of haptoglobin (1-1 and 2-1) provides evidence of a significant difference in their extent of glycosylation, with the glycan load of phenotype 2-1 being notably lighter. More detailed characterization of their glycosylation patterns is enabled by the recently introduced crosspath reactive chromatography (XP-RC) with on-line MS detection, a technique that combines chromatographic separation with in-line reduction of disulfide bonds to generate metastable haptoglobin subunits. Application of XP-RC to both haptoglobin phenotypes confirms that no modifications are present within their light chains, and provides a wealth of information on glycosylation patterns of the heavy chains. The haptoglobin 1-1 glycans are mature fully sialylated biantennary structures that exhibit high degrees of fucosylation. In contrast, phenotype 2-1 contains a significant fraction of incomplete biantennary structures and exhibit significantly lower levels of sialylation and fucosylation. The glycosylation patterns deduced from the XP-RC/MS measurements are in agreement with the conclusions of haptoglobin analysis by limited charge reduction, suggesting that the latter can be employed in situations when a fast assessment of a protein heterogeneity is needed (e.g., comparability studies of biopharmaceutical products). <br>

scholarly journals Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches

2021 ◽  
Author(s):  
Yang Yang ◽  
Jake W. Pawlowski ◽  
Ian J. Carrick ◽  
Igor Kaltashov
Keyword(s):  
Disulfide Bonds ◽  
Heterogeneous Systems ◽  
Structural Diversity ◽  
Mass Range ◽  
Protein Characterization ◽  
Charge Reduction ◽  
Average Mass ◽  
Significant Difference ◽  
Intact Mass

Intact-mass measurements are becoming an increasingly popular in mass spectrometry (MS) based protein characterization, as they allow the entire complement of proteoforms to be evaluated within a relatively short time. However, applications of this approach are currently limited to systems exhibiting relatively modest degrees of structural diversity, as the high extent of heterogeneity frequently prevents straightforward MS measurements. Incorporation of limited charge reduction into electrospray ionization (ESI) MS measurements provides an elegant way to obtain meaningful information on most heterogeneous systems, yielding not only the average mass of the protein, but also the mass range populated by various proteoforms. Application of this approach to characterization of two different phenotypes of haptoglobin (1-1 and 2-1) provides evidence of a significant difference in their extent of glycosylation, with the glycan load of phenotype 2-1 being notably lighter. More detailed characterization of their glycosylation patterns is enabled by the recently introduced crosspath reactive chromatography (XP-RC) with on-line MS detection, a technique that combines chromatographic separation with in-line reduction of disulfide bonds to generate metastable haptoglobin subunits. Application of XP-RC to both haptoglobin phenotypes confirms that no modifications are present within their light chains, and provides a wealth of information on glycosylation patterns of the heavy chains. The haptoglobin 1-1 glycans are mature fully sialylated biantennary structures that exhibit high degrees of fucosylation. In contrast, phenotype 2-1 contains a significant fraction of incomplete biantennary structures and exhibit significantly lower levels of sialylation and fucosylation. The glycosylation patterns deduced from the XP-RC/MS measurements are in agreement with the conclusions of haptoglobin analysis by limited charge reduction, suggesting that the latter can be employed in situations when a fast assessment of a protein heterogeneity is needed (e.g., comparability studies of biopharmaceutical products). <br>

Isolation and characterization of a new agglutinin from the red marine alga Hypnea cervicornis J. Agardh

2006 ◽  
Vol 84 (1) ◽  
pp. 49-54 ◽  
Author(s):  
K S Nascimento ◽  
C S Nagano ◽  
E V Nunes ◽  
R F Rodrigues ◽  
G V Goersch ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Marine Alga ◽  
Biochemical Characterization ◽  
Atlantic Coast ◽  
Haemagglutination Inhibition ◽  
Structural Diversity ◽  
Hypnea Musciformis ◽  
Isolation And Characterization ◽  
Red Marine Alga

The biochemical characterization of a new lectin (Hypnea cervicornis agglutinin or HCA) isolated from the Brazilian red alga H. cervicornis is reported. The haemagglutinating activity of the lectin was only inhibited by the glycoprotein porcine stomach mucin at a minimum inhibitory concentration of 19 µg·mL–1. No haemagglutination inhibition was detected after the addition of simple sugars. The MALDI-TOF molecular masses of native and reduced and carbamidomethylated HCA were, respectively, 9196.6 Da and 9988.2 Da, indicating that the primary structure of the protein is crosslinked by 7 disulfide bonds. This unusual structural feature among lectins, along with its N-terminal sequence and amino-acid composition, clearly shows that HCA belongs to a protein family distinct from the isolectins Hypnin A1 and A2 isolated from the related Japanese alga Hypnea japonica. On the other hand, HCA displayed a high degree of similarity to the agglutinin from the Brazilian species Hypnea musciformis. Our data indicate the occurrence of structural diversity among lectins of closely related species living in distant ecosystems, i.e., the Pacific coast of Japan and the Atlantic coast of Brazil, and support the hypothesis that the lectin content (lectinome) might serve as a biomarker for taxonomical purposes.Key words: agglutinin, lectin, isolation, Hypnea cervicornis, red marine alga.

Caracterização da Peneira Média em Clones de Coffea canephora

REVISTA FIMCA ◽  
2018 ◽  
Vol 5 (2) ◽  
pp. 28-31
Author(s):  
Darlan Darlan Sanches Barbosa Alves ◽  
Victor Mouzinho Spinelli ◽  
Marcos Santana Moraes ◽  
Carolina Augusto De Souza ◽  
Rodrigo da Silva Ribeiro ◽  
...  
Keyword(s):  
Genetic Improvement ◽  
Block Design ◽  
Coffea Canephora ◽  
Coffee Production ◽  
Randomized Block Design ◽  
Significant Difference ◽  
The Mean ◽  
Four Blocks ◽  
Selection Of

Introdução: O estado de Rondônia se destaca como tradicional produtor de café, sendo o segundo maior produtor brasileiro de C. canephora. No melhoramento genético de C. canephora, a seleção de plantas de elevada peneira média está associada à bebida de qualidade superior. Objetivos: O objetivo desse estudo foi avaliar a variabilidade genética de clones de C. canephora para o tamanho dos grãos, mensurado a partir da avaliação da peneira média (PM). Materiais e Métodos: Para isso, foi conduzido ao longo de dois anos agrícolas experimento no campo experimental da Embrapa no município de Ouro Preto do Oeste-RO, para a avaliação da peneira média de 130 genótipos (clones) com características das variedades botânicas Conilon, Robusta e híbridos intervarietais. O delineamento experimental utilizado foi de blocos ao acaso, com quatro repetições de quatro plantas por parcela. Resultados: Não houve resultados significativos para a interação clones X anos, indicando uma maior consistência no comportamento das plantas ao longo do tempo. Porém foram observadas diferenças significativas para o tamanho dos grãos entre os genótipos avaliados, possibilitando selecionar genótipos superiores. Conclusão: Os genótipos agruparam-se em cinco classes de acordo com o teste de média, subsidiando a caracterização de um gradiente de variabilidade da característica avaliada ABSTRACTIntroduction: Coffea canephora accounts for approximately 35% of the world's coffee production. The state of Rondônia stands out as a traditional coffee producer, being the second largest Brazilian producer of C. canephora. In the classical genetic improvement of C. anephora, the selection of plants of high average sieve is associated with a drink of superior quality. Objectives: The objective of this udy was to evaluate the genetic variability of Coffea canephora clones for the agronomic medium sieve (PM). Materials and Methods: The experiment was conducted in the experimental field of Embrapa, municipality of OuroPreto do Oeste-RO, located at coordinates 10º44'53 "S and 62º12'57". One hundred thirty genotypes (clones) of botanical characteristics Conilon, Robusta and intervarietal hybrids were evaluated in the agricultural years 2013-2014 and 2014-2015. The experimental design was a randomized block design with four blocks and four plants per plot, spacing 3.5 x 1.5 meters between plants. Results: Significant difference was found for the grain size. According to the F test, at 5% probability, the genotypes were grouped into five classes according to the mean test. Conclusion: The results obtained subsidized the characterization of a variability gradient of the evaluated trait.

scholarly journals A global non-invasive methodology for the phenotyping of potato under water deficit conditions using imaging, physiological and molecular tools

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
M. Musse ◽  
G. Hajjar ◽  
N. Ali ◽  
B. Billiot ◽  
G. Joly ◽  
...  
Keyword(s):  
Water Stress ◽  
Water Deficit ◽  
Soil Conditions ◽  
Growth Stages ◽  
Dry Matter Content ◽  
Tuber Growth ◽  
Non Invasive ◽  
Potato Plants ◽  
Significant Difference

Abstract Background Drought is a major consequence of global heating that has negative impacts on agriculture. Potato is a drought-sensitive crop; tuber growth and dry matter content may both be impacted. Moreover, water deficit can induce physiological disorders such as glassy tubers and internal rust spots. The response of potato plants to drought is complex and can be affected by cultivar type, climatic and soil conditions, and the point at which water stress occurs during growth. The characterization of adaptive responses in plants presents a major phenotyping challenge. There is therefore a demand for the development of non-invasive analytical techniques to improve phenotyping. Results This project aimed to take advantage of innovative approaches in MRI, phenotyping and molecular biology to evaluate the effects of water stress on potato plants during growth. Plants were cultivated in pots under different water conditions. A control group of plants were cultivated under optimal water uptake conditions. Other groups were cultivated under mild and severe water deficiency conditions (40 and 20% of field capacity, respectively) applied at different tuber growth phases (initiation, filling). Water stress was evaluated by monitoring soil water potential. Two fully-equipped imaging cabinets were set up to characterize plant morphology using high definition color cameras (top and side views) and to measure plant stress using RGB cameras. The response of potato plants to water stress depended on the intensity and duration of the stress. Three-dimensional morphological images of the underground organs of potato plants in pots were recorded using a 1.5 T MRI scanner. A significant difference in growth kinetics was observed at the early growth stages between the control and stressed plants. Quantitative PCR analysis was carried out at molecular level on the expression patterns of selected drought-responsive genes. Variations in stress levels were seen to modulate ABA and drought-responsive ABA-dependent and ABA-independent genes. Conclusions This methodology, when applied to the phenotyping of potato under water deficit conditions, provides a quantitative analysis of leaves and tubers properties at microstructural and molecular levels. The approaches thus developed could therefore be effective in the multi-scale characterization of plant response to water stress, from organ development to gene expression.

scholarly journals Crystallization of Random Metallocene-Catalyzed Propylene-Based Copolymers with Ethylene and 1-Hexene on Rapid Cooling

Polymers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 2091
Author(s):  
Daniela Mileva ◽  
Jingbo Wang ◽  
René Androsch ◽  
Katalee Jariyavidyanont ◽  
Markus Gahleitner ◽  
...  
Keyword(s):  
Mechanical Performance ◽  
Metallocene Catalyst ◽  
Nucleating Agent ◽  
Random Copolymers ◽  
Rapid Cooling ◽  
Significant Difference ◽  
Chip Calorimetry ◽  
High Cooling Rates ◽  
Fast Scanning Chip Calorimetry

Propylene-based random copolymers with either ethylene or 1-hexene as comonomer, produced using a metallocene catalyst, were studied regarding their crystallization behaviors, with a focus on rapid cooling. To get an impression of processing effects, fast scanning chip calorimetry (FSC) was used in addition to the characterization of the mechanical performance. When comparing the comonomer type and the relation to commercial grades based on Ziegler–Natta-type catalysts, both an interaction with the catalyst-related regio-defects and a significant difference between ethylene and 1-hexene was observed. A soluble-type nucleating agent was found to modify the behavior, but to an increasingly lesser degree at high cooling rates.

scholarly journals Integration of Dental Implants in Conjunction with EDTA-Conditioned Dentin Grafts: An Experimental Study

2021 ◽  
Vol 9 (6) ◽  
pp. 63
Author(s):  
Payam Farzad ◽  
Ted Lundgren ◽  
Adel Al-Asfour ◽  
Lars Andersson ◽  
Christer Dahlin
Keyword(s):  
Direct Contact ◽  
Mineral Content ◽  
X Rays ◽  
X Ray ◽  
Dentin Surface ◽  
Significant Difference ◽  
C And N ◽  
Micro Implants ◽  
Demineralized Dentin

This study was undertaken to investigate the integration of titanium micro-implants installed in conjunction with previously dentin-grafted areas and to study the morphological appearance, mineral content, and healing pattern of xenogenic EDTA-conditioned dentin blocks and granules grafted to cavities in the tibial bone of rabbits. Demineralized and non-demineralized dentin blocks and granules from human premolars were implanted into cavities prepared on the lateral aspects of the tibias of rabbits. After a healing period of six months, micro-implants were installed at each surgical site. Histological examinations were carried out after 24 weeks. Characterization of the EDTA-conditioned dentin blocks was performed by means of light microscopy, dental X-rays, scanning electron microscopy, and energy dispersive X-ray analysis (EDX). No implants were found to be integrated in direct contact with the dentin particles or blocks. On the EDTA-conditioned dentin surface, the organic marker elements C and N dominated, as revealed by EDX. The hydroxyapatite constituents Ca and P were almost absent on the dentin surface. No statistically significant difference was observed between the EDTA-conditioned and non-demineralized dentin, as revealed by BIC and BA. The bone-inductive capacity of the dentin material seemed limited, although demineralization by means of EDTA indicated higher BIC and BA values in conjunction with the installed implants in the area. A 12 h EDTA treatment did not fully decalcify the grafts, as revealed by X-ray analysis.

scholarly journals Design of a force-measuring setup for colorectal compression anastomosis and first ex-vivo results

2021 ◽  
Author(s):  
Jana Steger ◽  
Isabella Patzke ◽  
Maximilian Berlet ◽  
Stefanie Ficht ◽  
Markus Eblenkamp ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Tissue Fixation ◽  
Force Transmission ◽  
Tissue Thickness ◽  
Colon Tissue ◽  
Device Development ◽  
Compression Anastomosis ◽  
Technical Requirements ◽  
Significant Difference

Abstract Purpose The introduction of novel endoscopic instruments is essential to reduce trauma in visceral surgery. However, endoscopic device development is hampered by challenges in respecting the dimensional restrictions, due to the narrow access route, and by achieving adequate force transmission. As the overall goal of our research is the development of a patient adaptable, endoscopic anastomosis manipulator, biomechanical and size-related characterization of gastrointestinal organs are needed to determine technical requirements and thresholds to define functional design and load-compatible dimensioning of devices. Methods We built an experimental setup to measure colon tissue compression piercing forces. We tested 54 parameter sets, including variations of three tissue fixation configurations, three piercing body configurations (four, eight, twelve spikes) and insertion trajectories of constant velocities (5 mms−1, 10 mms−1,15 mms−1) and constant accelerations (5 mms−2, 10 mms−2, 15 mms−2) each in 5 samples. Furthermore, anatomical parameters (lumen diameter, tissue thickness) were recorded. Results There was no statistically significant difference in insertion forces neither between the trajectory groups, nor for variation of tissue fixation configurations. However, we observed a statistically significant increase in insertion forces for increasing number of spikes. The maximum mean peak forces for four, eight and twelve spikes were 6.4 ± 1.5 N, 13.6 ± 1.4 N and 21.7 ± 5.8 N, respectively. The 5th percentile of specimen lumen diameters and pierced tissue thickness were 24.1 mm and 2.8 mm, and the 95th percentiles 40.1 mm and 4.8 mm, respectively. Conclusion The setup enabled reliable biomechanical characterization of colon material, on the base of which design specifications for an endoscopic anastomosis device were derived. The axial implant closure unit must enable axial force transmission of at least 28 N (22 ± 6 N). Implant and applicator diameters must cover a range between 24 and 40 mm, and the implant gap, compressing anastomosed tissue, between 2 and 5 mm.

scholarly journals Odorant-binding protein. Characterization of ligand binding.

1990 ◽  
Vol 265 (11) ◽  
pp. 6118-6125
Author(s):  
J Pevsner ◽  
V Hou ◽  
A M Snowman ◽  
S H Snyder
Keyword(s):  
Ligand Binding ◽  
Binding Protein ◽  
Protein Characterization ◽  
Odorant Binding Protein ◽  
Odorant Binding

scholarly journals Unravelling the Skin Secretion Peptides of the Gliding Leaf Frog, Agalychnis spurrelli (Hylidae)

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 667 ◽  
Author(s):  
Carolina Proaño-Bolaños ◽  
Ailín Blasco-Zúñiga ◽  
José Rafael Almeida ◽  
Lei Wang ◽  
Miguel Angel Llumiquinga ◽  
...  
Keyword(s):  
Inhibitory Concentration ◽  
Structural Diversity ◽  
Biologically Active ◽  
Biological Characterization ◽  
Skin Secretion ◽  
Peptide Profile ◽  
Active Chemical ◽  
Molecular Masses ◽  
Skin Secretions

Frog skin secretions contain medically-valuable molecules, which are useful for the discovery of new biopharmaceuticals. The peptide profile of the skin secretion of Agalychnis spurrelli has not been investigated; therefore, the structural and biological characterization of its compounds signify an inestimable opportunity to acquire new biologically-active chemical scaffolds. In this work, skin secretion from this amphibian was analysed by molecular cloning and tandem mass spectrometry. Although the extent of this work was not exhaustive, eleven skin secretion peptides belonging to five peptide families were identified. Among these, we report the occurrence of two phyllokinins, and one medusin-SP which were previously reported in other related species. In addition, eight novel peptides were identified, including four dermaseptins, DRS-SP2 to DRS-SP5, one phylloseptin-SP1, and three orphan peptides. Phylloseptin-SP1 and dermaseptins-SP2 were identified in HPLC fractions based on their molecular masses determined by MALDI-TOF MS. Among the antimicrobial peptides, dermaseptin-SP2 was the most potent, inhibiting Escherichia coli, Staphylococcus aureus, and ORSA with a minimum inhibitory concentration (MIC) of 2.68 μM, and Candida albicans with an MIC of 10.71 μM, without haemolytic effects. The peptides described in this study represent but a superficial glance at the considerable structural diversity of bioactive peptides produced in the skin secretion of A. spurrelli.

Sign in / Sign up

Export Citation Format

Share Document