Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC–MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing ...number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 μg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 μg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.
Affinity chromatography is a powerful technology for phosphopeptide enrichment from body fluids. Saliva is a non-invasive body fluid for disease diagnosis, while few studies applied affinity ...enrichment for saliva phosphoproteome. In this study, we tested two kinds of affinity chromatography materials, Ti4+-IMAC (immobilized metal affinity chromatography) and CaTiO3, for the enrichment of phosphopeptides. Through comparison, Ti4+-IMAC method was demonstrated as the superior one, which was utilized for the comprehensive analysis of salivary phosphoproteome. More than 360 phosphoproteins were specifically extracted and identified from human saliva. Ti4+-IMAC method was further applied to compare the phosphoprotein profiling in the saliva of lung cancer group and normal control group through label-free quantification. Accordingly, 477 and 699 phosphopeptides were enriched, respectively, which corresponded to 339 and 466 proteins. In total, 796 unique phosphopeptides were revealed for 517 saliva phosphoproteins. In particular, 709 phosphorylation sites were identified, among which 26 were up-regulated (>1.5) and 149 were down-regulated (<0.66) in lung cancer. Their corresponding proteins were mainly associated with cancer promotion, system disorder, and organismal injury. Our data collectively demonstrated that salivary phosphopeptides can be comprehensively characterized through Ti4+-IMAC method. These discovered phosphoprotein candidates might be used for lung cancer detection through salivary diagnostics.
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•Ti4+-affinity chromatography showed superior phosphopeptide enrichment efficiency.•517 saliva phosphoproteins with 709 phosphosites were identified and quantified.•Dysregulated saliva phosphoproteins and phosphosites were revealed for lung cancer.
•Printed monolith adsorption uses a 3D-printed monolithic structure for chromatography.•PMA technology combined with an immobilized metal affinity ligand was used to purify polyhistidine-tagged ...proteins.•Single-step protein purification using IMAC-functionalized PMA columns was demonstrated.•The IMAC-functionalized PMA columns reduce purification time and costs during the purification process.
Bio-separation is a crucial process in biotechnology and biochemical engineering for separating biological macromolecules, and the field has long relied on bead-based and expanded bed chromatography. Printed monolith adsorption (PMA) is a new alternative to which uses a 3D-printed monolithic structure containing self-supporting, ordered flow channels. PMA allows for direct purification of biological molecules from crude cell lysates and cell cultures, and like the other technologies, can functionalized to specifically target a molecule and enable affinity chromatography. Here we have combined PMA technology with an immobilized metal affinity ligand (iminodiacetic acid) to provide selectivity of binding to polyhistidine-tagged proteins during PMA chromatography. Two different PMA structures were created and tested for both static and dynamic protein-binding capacity. At comparative linear flow rates, the dynamic binding capacity of both columns was ≈3 mg/mL, while static capacity was shown to differentiate based on column voidage. We show that a polyhistidine-tagged protein can be directly purified from crude lysate with comparable results to the available commercial providers of IMAC, and with a substantially reduced purification time.
In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains ...with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize–stabilize–purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review.
•Use of immobilized antibodies to get the one step immobilization–purification•Use of domains with specific affinity for some ligands to immobilize/purify tagged proteins•Immobilization/purification by using of domains that increase the enzyme affinity for standard supports•Glyoxyl supports and one step immobilization–purification of multimeric proteins•Medium engineering and standard supports for immobilization/purification•Tailor made heterofunctional supports for large protein immobilization/purification
Phosphopeptides were of great significance in disease diagnosis and monitoring its dynamic changes. In this article, we proposed a more efficient method to synthesize a kind of bimetallic mesoporous ...silica nanomaterials (Fe3O4@mSiO2-PO3-Ti4+/Zr4+) and applied it to the analysis of phosphopeptides in human saliva samples based on IMAC technology. The chelation group was introduced into mesopores at the same time as the formation of mesoporous silica which significantly reduced the synthesis procedure and improved the synthesis efficiency. The as-prepared materials showed great sensitivity, selectivity and size-exclusion performance for phosphopeptides in standard β-casein digests. More importantly, the materials identified 85 phosphopeptides in disease saliva samples which provided a candidate choice in future clinical examination.
A magnetic bimetallic platform combining IMAC and mesoporous silica was established to specifically identify phosphopeptides in human saliva. Display omitted
•Introduced chelation group into mesopores through one-pot reaction.•Reduced synthesis procedures and provided great potential in future commercial application.•Established a bimetallic platform for identification of phosphopeptides in human saliva samples.
The Chimera antigen receptor (CAR)-T cell therapy has gained great success in the clinic. However, there are still major challenges for its wider applications in a variety of cancer types including ...lack of effectiveness due to the highly complex tumor microenvironment, and the forbiddingly high cost due to the personalized manufacturing procedures. In order to overcome these hurdles, numerous efforts have been spent focusing on optimizing Chimera antigen receptors, engineering and improving T cell capacity, exploiting features of subsets of T cell or NK cells, or making off-the-shelf universal cells. Here, we developed induced pluripotent stem cells (iPSCs)-derived, CAR-expressing macrophage cells (CAR-iMac). CAR expression confers antigen-dependent macrophage functions such as expression and secretion of cytokines, polarization toward the pro-inflammatory/anti-tumor state, enhanced phagocytosis of tumor cells, and in vivo anticancer cell activity. This technology platform for the first time provides an unlimited source of iPSC-derived engineered CAR-macrophage cells which could be utilized to eliminate cancer cells.
The work describes the preparation of a new magnetic phase for batch enrichment of phosphopeptides. The material exploits the advantages of magnetic solid phase extraction and couples them with the ...most employed approach for phosphopeptide enrichment, i.e. Ti4+-IMAC. In order to immobilize Ti4+ ions on the surface of the magnetite nanoparticles, they were first covered by a silica shell and then modified to expose at the surface bromine containing groups. Glycidyl methacrylate was subsequently polymerized from these groups using the “grafting from” approach by the activator regenerated by electron transfer–atom transfer radical polymerization (ARGET-ATRP) technique. Finally, the glycidyl groups were reacted with iminodiacetic acid to functionalize the material with moieties suitable for coordination. The prepared material was extensively characterized and subsequently tested for enrichment of a bovine serum albumin mixture with casein to ascertain its potential. With positive results, the new magnetic polymeric material was further employed to set up an enrichment method on yeast protein digest based on shotgun proteomics. The sample to phase ratio was optimized and the best condition compared to a commercial TiO2 spin column. At the end of the comparison, the new material proved better and could enrich a larger total number of phosphopeptides with increased selectivity. All these conclusions and the test performed on a real complex sample within the final shotgun application further support the applicability of the new material in phosphopeptide analysis of real matrices.
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•ARGET-ATRP grafting from was exploited to prepare a new functional polymer for IMAC.•Magnetic extraction method for phosphopeptides enrichment in yeast digest.•Average intensity twice larger for phosphopeptide than non-phosphorylated peptides.•Shotgun phosphoproteomics allows a systematic comparison to commercial systems.•Fe3O4@silica@GMA@IDA@Ti4+ outperforms TiO2 spin column enrichment.
Indian residences are vulnerable to heat-driven discomfort amid the mounting prevalence of weather extremes, residential design and construction practices, and densifying urbanscapes. Therefore, it ...is vital to understand the thermal comfort characteristics of nationwide residences. This study proposes an adaptive thermal comfort model based on yearlong field surveys in eight cities located across five climate zones of India – the India Model for Adaptive Comfort - Residential (IMAC-R). The model prescribes the operative temperature bands for 80% and 90% thermal acceptability in correlation with the outdoor reference temperature, applicable to mixed-mode (MM) and naturally ventilated (NV) residences.
More than 80% of the Indian residential occupants experienced a neutral thermal sensation in the indoor operative range of 16.3–35 °C in response to a 5.5–33 °C variation in the 30-day outdoor running mean temperature. Comparing the proposed model with the PMV model revealed that the latter underpredicts the thermal adaptivity of Indian occupants. The model was also compared against its predecessor – India Model for Adaptive Comfort for Commercial Buildings (IMAC MM and NV), along with relevant global and regional thermal comfort models. On average, the neutral temperature prescribed by IMAC-R was warmer than the temperatures prescribed by IMAC MM and NV by 2.9 °C and 2.1 °C, respectively; it was also warmer than the temperature prescribed by the recent ASHRAE-55 and EN 16798-1 models by 2 °C and 0.3 °C, respectively. IMAC-R reserves the prospect of addressing the thermal comfort needs of the national population while paving the way for long-term energy savings and climate action.
•An adaptive model is proposed for mixed-mode and naturally ventilated residences across the Indian climates.•Indian residential occupants are more adaptive than as predicted by ASHRAE-55, EN 15251, and PMV models.•Fan/window operation and change in clothing insulation were the key adaptive responses.•Comfort modelling is explored in correlation with Universal Thermal Climate Index.
•Ni2+-O-CMCS-CCs was synthesized for lysozyme purification.•Adsorption of 244.6 and 15.3 mg/g was reached with Ni2+ and without Ni2+ particles.•More than 95% of adsorbed lysozyme was desorbed in ...about 20 min.•The same column was showed 35 adsorption-desorption cycles.
We synthesized Ni2+-attached O-Carboxymethyl chitosan Schiff base complexes embedded composite cryogels (Ni2+-O-CMCS-CCs) by means of polymerization of gel-forming precursors at subzero temperatures. Prepared affinity cryogel showed excellent adsorption performance for lysozyme selected as model protein to test adsorption parameters, demonstrating an adsorption capacity of 244.6 mg/g (15.3 mg/g for Ni2+ minus O-CMCS-CCs), with fast adsorption equilibrium within 30 min and good reversibility. The performance of Ni2+-O-CMCS-CCs for lysozyme was also evaluated by SDS-PAGE, and a purification efficiency of 86.9% with 89.5% purification yield was determined. The swelling test, FT-IR, and SEM analysis were carried out for the characterization of Ni2+-O-CMCS-CCs. At the end of 35 adsorption-desorption cycles, there was no significant change in the adsorption capacity.
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