Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization

Infrared Multiple-Photon Dissociation Action Spectroscopy of the b(2)(+) Ion from PPG: Evidence of Third Residue Affecting b(2)(+) Fragment Structure

Infrared multiple-photon dissociation (IRMPD) action spectroscopy was performed on the b2 + fragment ion from the protonated PPG tripeptide. Comparison of the experimental infrared spectrum with computed spectra for both oxazolone and diketopiperazine structures indicates that the majority of the fragment ion population has an oxazolone structure with the remainder having a diketopiperazine structure. This result is in contrast with a recent study of the IRMPD action spectrum of the PP b2 + fragment ion from PPP, which was found to be nearly 100% diketopiperazine (Martens et al. Int. J. Mass Spectrom. 2015, 377, 179). The diketopiperazine b2 + ion is thermodynamically more stable than the oxazolone but normally requires a trans/cis peptide bond isomerization in the dissociating peptide. Martens et al. showed through IRMPD action spectroscopy that the PPP precursor ion was in a conformation in which the first peptide bond is already in the cis conformation and thus it was energetically favorable to form the thermodynamically-favored diketopiperazine b2 + ion. In the present case, solution-phase NMR spectroscopy and gas-phase IRMPD action spectroscopy show that the PPGprecursor ion has its first amide bond in a trans configuration suggesting that the third residue is playing an important role in both the structure of the peptide and the associated ring-closure barriers for oxazolone and diketopiperazine formation

Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization

De novo design of protein logic gates

The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo–designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the TIM3 gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions

Ni

Supercapacitors have become a research hotspot in the field of energy storage due to their highpower density, fast charging/discharging ability, long-term stability and safety. Nevertheless, relatively low energy density hindered their application. Herein, Ni3S2/Zn0.76Co0.24S (NZCS) microsphere were synthesized using a facile two-step hydrothermal process. The polymetallic synergies can improve conductivity and shorten ion transport path. The uniform particle distribution provided numerous active sites for faradaic reactions. The NZCS mircosphere showed a large capacity of 571.5 C g-1 at 1 A g-1 and 87% rate rentention when the current increases by 10 times. A hybrid supercapacitor assmebled by NZCS cathode and active carbon anode demostrate a high energy density of 44.1 Wh kg-1 (407.0 W kg-1) and a stable cycliability of 15,000 cycles with 15% loss. Thus, NZCS is a promising electrode material for high performance supercapacitor

Synergistyczny wpływ obróbki plazmowej i struktury tkaniny na parametry odporności na przebicie tkanin kompozytowych STF/aramid

In order to improve the contribution of STF and fabric to the stab resistance of STF-impregnated aramid soft armour materials, the plasma treatment of various fabric structures was conducted. This study explored the interactive effects of plasma treatment, fabric structure and particle size on the spike and knife resistance properties of plasma-treated STF/Aramid fabrics. Fumed silica and polyethylene glycol (PEG) based STFs were prepared with various particle sizes (15 nm, 75 nm) at a solid content of 15%. Various weave structures of fabrics (plain, 2/2 twill, 5/3 satin, 2/2 basket) were impregnated with STF and then plasma-treatment conducted. The rheological behaviour of STF in various silica sizes as well as the spike and knife quasi-static stab resistances of the resultant plasma-treated STF/aramid fabrics in various weave structures were both explored. The results show that the various weave structures of STF/Aramid fabrics treated with plasma exhibited a significant enhancement of quasi-static spike resistance. Furthermor, 2/2 twill, 5/3 satin and basket weaving plasma-treated STF/Aramid with a coarser silica particle in STF showed a higher improvement in quasi-static spike resistance. Interactive effect results show that the plasma treatment of fabric and the silica size in STF affected spike resistance more significantly, while knife resistance was only significantly affected by the fabric structure.Aby poprawić odporność na przebicie tkaniny kompozytowych STF/aramid przeprowadzono ich obróbkę plazmową. Zbadano interaktywny wpływ obróbki plazmowej, struktury tkaniny i wielkości cząstek na właściwości odporności na przebicie kolcami i nożem tkanin kompozytowych STF/aramid poddanych działaniu plazmy. Przygotowano STF na bazie zmatowionej krzemionki koloidalnej i glikolu polietylenowego (PEG) o różnych wielkościach cząstek (15 i 75 nm) o zawartości substancji stałych wynoszącej 15%. Różne struktury splotu tkanin (m.in. gładkie, 2/2 diagonalne i 5/3 satynowe) zostały zaimpregnowane STF, a następnie przeprowadzono obróbkę plazmą. Badano zarówno zachowanie reologiczne w przypadku różnych rozmiarów krzemionki, jak i quasi-statyczne odporności na przebicie kolcami i nożem otrzymanych tkanin STF/aramid poddanych obróbce plazmowej i wykonanych z zastosowaniem różnych struktur splotu. Wyniki pokazały, że różne struktury splotu tkanin STF aramid poddanych obróbce plazmą wykazały znaczne zwiększenie quasi-statycznej odporności na przebicie kolcami. Ponadto tkany z grubszą cząstką krzemionki w STF wykazały poprawę w quasi-statycznej odporności na przebicie kolcami. Wyniki pokazały, że obróbka plazmowa tkaniny i rozmiar krzemionki w STF wpłynęły bardziej na odporność na przebicie kolcami, podczas gdy na wytrzymałość na przebicie nożem istotny wpływ miała tylko struktura tkaniny

Development and validation of prognostic nomograms for large hepatocellular carcinoma after HAIC

Background and aims: Hepatic arterial infusion chemotherapy (HAIC) using the FOLFOX regimen (oxaliplatin plus fluorouracil and leucovorin) is a promising option for large hepatocellular carcinoma (HCC). However, post-HAIC prognosis can vary in different patients due to tumor heterogeneity. Herein, we established two nomogram models to assess the survival prognosis of patients after HAIC combination therapy. Methods: A total of 1082 HCC patients who underwent initial HAIC were enrolled between February 2014 and December 2021. We built two nomogram models for survival prediction: the preoperative nomogram (pre-HAICN) using preoperative clinical data and the postoperative nomogram (post-HAICN) based on pre-HAICN and combination therapy. The two nomogram models were internally validated in one hospital and externally validated in four hospitals. A multivariate Cox proportional hazards model was used to identify risk factors for overall survival (OS). The performance outcomes of all models were compared by area under the receiver operating characteristic curve (AUC) analysis with the DeLong test. Results: Multivariable analysis identified larger tumor size, vascular invasion, metastasis, high albumin–bilirubin grade, and high alpha-fetoprotein as indicators for poor prognosis. With these variables, the pre-HAICN provided three risk strata for OS in the training cohort: low risk (5-year OS, 44.9%), middle risk (5-year OS, 20.6%), and high risk (5-year OS, 4.9%). The discrimination of the three strata was improved significantly in the post-HAICN, which included the above-mentioned factors and number of sessions, combination with immune checkpoint inhibitors, tyrosine kinase inhibitors, and local therapy (AUC, 0.802 versus 0.811, p  < 0.001). Conclusions: The nomogram models are essential to identify patients with large HCC suitable for treatment with HAIC combination therapy and may potentially benefit personalized decision-making. Lay summary Hepatic arterial infusion chemotherapy (HAIC) provides sustained higher concentrations of chemotherapy agents in large hepatocellular carcinoma (HCC) by hepatic intra-arterial, result in better objective response outperformed the intravenous administration. HAIC is significantly correlated with favorable survival outcome and obtains extensive support in the effective and safe treatment of intermediate advanced-stage HCC. In view of the high heterogeneity of HCC, there is no consensus regarding the optimal tool for risk stratification before HAIC alone or HAIC combined with tyrosine kinase inhibitors or immune checkpoint inhibitors treatment in HCC. In this large collaboration, we established two nomogram models to estimate the prognosis and evaluate the survival benefits with different HAIC combination therapy. It could help physicians in decision-making before HAIC and comprehensive treatment for large HCC patients in clinical practice and future trials

Diffusion Restriction in the Splenium: A Comparative Study of Cytotoxic Lesions of the Corpus Callosum (CLOCCs) versus Lesions of Vascular Etiology

Cytotoxic lesions of the corpus callosum (CLOCCs) have broad differential diagnoses. Differentiating these lesions from lesions of vascular etiology is of high clinical significance. We compared the clinical and radiological characteristics and outcomes between vascular splenial lesions and CLOCCs in a retrospective cohort study. We examined the clinical and radiologic characteristics and outcomes in 155 patients with diffusion restriction in the splenium of the corpus callosum. Patients with lesions attributed to a vascular etiology (N = 124) were older (64.1 vs. 34.6 years old, p 1 vascular risk factor (91.1% vs. 45.2%, p p ≤ 0.05). CLOCCs (N = 31) more commonly had midline splenial involvement (p p p = 0.002). The rate of in-hospital mortality was significantly higher in patients with vascular etiology lesions (p = 0.04). Across vascular etiology lesions, cardio-embolism was the most frequent stroke mechanism (29.8%). Our study shows that corpus callosum diffusion restricted lesions of vascular etiology and CLOCCs are associated with different baseline, clinical, and radiological characteristics and outcomes. Accurately differentiating these lesions is important for appropriate treatment and secondary prevention

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization