Online comprehensive two-dimensional liquid chromatography (LC × LC) offers ways to achieve high-performance separations in terms of peak capacity (exceeding 1000) and additional selectivity to ...realize applications that cannot be addressed with one-dimensional chromatography (1D-LC). However, the greater resolving power of LC × LC comes at the price of higher dilutions (thus, reduced sensitivity) and, often, long analysis times (>100 min). The need to preserve the separation attained in the first dimension (1D) causes greater dilution for LC × LC, in comparison with 1D-LC, and long analysis times to sample the 1D with an adequate number of second dimension separations. A way to significantly reduce these downsides is to introduce a concentration step between the two chromatographic dimensions. In this work we present a possible active-modulation approach to concentrate the fractions of 1D effluent. A typical LC × LC system is used with the addition of a dilution flow to decrease the strength of the 1D effluent and a modulation unit that uses trap columns. The potential of this approach is demonstrated for the separation of tristyrylphenol ethoxylate phosphate surfactants, using a combination of hydrophilic interaction and reversed-phase liquid chromatography. The modified LC × LC system enabled us to halve the analysis time necessary to obtain a similar degree of separation efficiency with respect to UHPLC based LC × LC and of 5 times with respect to HPLC instrumentation (40 compared with 80 and 200 min, respectively), while at the same time reducing dilution (DF of 142, 299, and 1529, respectively) and solvent consumption per analysis (78, 120, and 800 mL, respectively).
SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity ...assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein's structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.
Online comprehensive two‐dimensional liquid chromatography has become an attractive option for the analysis of complex nonvolatile samples found in various fields (e.g. environmental studies, food, ...life, and polymer sciences). Two‐dimensional liquid chromatography complements the highly popular hyphenated systems that combine liquid chromatography with mass spectrometry. Two‐dimensional liquid chromatography is also applied to the analysis of samples that are not compatible with mass spectrometry (e.g. high‐molecular‐weight polymers), providing important information on the distribution of the sample components along chemical dimensions (molecular weight, charge, lipophilicity, stereochemistry, etc.). Also, in comparison with conventional one‐dimensional liquid chromatography, two‐dimensional liquid chromatography provides a greater separation power (peak capacity). Because of the additional selectivity and higher peak capacity, the combination of two‐dimensional liquid chromatography with mass spectrometry allows for simpler mixtures of compounds to be introduced in the ion source at any given time, improving quantitative analysis by reducing matrix effects. In this review, we summarize the rationale and principles of two‐dimensional liquid chromatography experiments, describe advantages and disadvantages of combining different selectivities and discuss strategies to improve the quality of two‐dimensional liquid chromatography separations.
Current approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they ...also result in a loss of valuable information regarding certain subclasses, allotypes and co-occuring post-translational modifications (PTMs). Unfortunately, the high variability of polyclonal IgGs makes their intact mass spectrometry (MS) analysis extremely challenging. We propose here a middle-up strategy for the analysis of the intact fragment crystallizable (Fc) region of human plasma IgGs, with the aim of acquiring integrated information of the
-glycosylation and other PTMs of subclasses and allotypes. Human plasma IgG was isolated using Fc-specific beads followed by an on-bead C
2 domain digestion with the enzyme IdeS. The obtained mixture of Fc subunits was analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) hyphenated with MS. CE-MS provided separation of different IgG-subclasses and allotypes, while HILIC-MS allowed resolution of the different glycoforms and their oxidized variants. The orthogonality of these techniques was key to reliably assign Fc allotypes. Five individual donors were analyzed using this approach. Heterozygosis was observed in all the analyzed donors resulting in a total of 12 allotypes identified. The assignments were further confirmed using recombinant monoclonal IgG allotypes as standards. While the glycosylation patterns were similar within allotypes of the same subclass, clear differences were observed between IgG subclasses and donors, highlighting the relevance of the proposed approach. In a single analysis, glycosylation levels specific for each allotype, relative abundances of subclasses and information on co-occurring modifications are obtained. This middle-up method represents an important step toward a comprehensive analysis of immunoglobulin G-Fc variants.
The transcription factor MYB plays a pivotal role in haematopoietic homoeostasis and its aberrant expression is involved in the genesis and maintenance of acute myeloid leukaemia (AML). We have ...previously demonstrated that not all AML subtypes display the same dependency on MYB expression and that such variability is dictated by the nature of the driver mutation. However, whether this difference in MYB dependency is a general trend in AML remains to be further elucidated. Here, we investigate the role of MYB in human leukaemia by performing siRNA-mediated knock-down in cell line models of AML with different driver lesions. We show that the characteristic reduction in proliferation and the concomitant induction of myeloid differentiation that is observed in MLL-rearranged and t(8;21) leukaemias upon MYB suppression is not seen in AML cells with a complex karyotype. Transcriptome analyses revealed that MYB ablation produces consensual increase of MAFB expression in MYB-dependent cells and, interestingly, the ectopic expression of MAFB could phenocopy the effect of MYB suppression. Accordingly, in silico stratification analyses of molecular data from AML patients revealed a reciprocal relationship between MYB and MAFB expression, highlighting a novel biological interconnection between these two factors in AML and supporting new rationales of MAFB targeting in MLL-rearranged leukaemias.
•The ongoing Corona Virus Disease 2019 (COVID–19) pandemic has proven to be an important psychosocial stressor for mental illness.•Purpose of the present manuscript is to assess the frequency and ...characteristics of patients with First Episodes Psychosis (FEP) hospitalized in the first four months since lockdown in Milan.•Patients with FEP in 2020 were significantly older than patients in 2019.•Substances abuse was statistically significant lower in patients with FEP in 2020 than in 2019.
The ongoing Corona Virus Disease 2019 (COVID–19) pandemic appears to increase risk for mental illness, either directly due to inflammation caused by the virus or indirectly due to related psychosocial stress, resulting in the development of both anxious-depressive and psychotic symptoms. The purpose of the present study was to assess the frequency and characteristics of all patients with First Episodes Psychosis (FEP) without COVID-19 infection hospitalized in the first four months since lockdown in Milan.
We recruited sixty-two patients hospitalized between March 8 to July 8, 2020 versus those
first hospitalized in the same period in 2019. The two subgroups were compared for sociodemographic variables and clinical characteristics of the episodes. Patients with FEP in 2020 were significantly older than patients with FEP in 2021, and presented with significantly less substances abuse.
Interestingly, patients presenting with FEP in 2020 were significantly older than patients with FEP in 2019. These data are compatible with the greater vulnerability to stressful factors during the pandemic, as well as with the greater concern regarding a possible COVID-19 infection producing brain damage causing the FEP.
We constructed a high-performance biosensor for detecting uric acid by immobilizing an engineered urate oxidase on gold nanoparticles deposited on a carbon-glass electrode. This biosensor showed a ...low limit-of-detection (9.16 nM), a high sensitivity (14 μA/μM), a wide range of linearity (50 nM-1 mM), and more than 28 days lifetime.
Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. ...However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA may cause ionization suppression and adduct formation, leading to reduced analyte sensitivity. To address this, we describe a membrane-based microfluidic chip with multiple parallel channels for the selective post-column removal of TFA anions from HILIC. An anion-exchange membrane was used to physically separate the column effluent from a stripper flow solution comprising acetonitrile, formic acid, and propionic acid. The exchange of ions allowed the post-column removal of TFA used during HILIC separation of model proteins. The multichannel design of the device allows the use of flow rates of 0.2 mL/min without the need for a flow splitter, using mobile phases containing 0.1% TFA (13 mM). Separation selectivity and efficiency were maintained (with minor band broadening effects) while increasing the signal intensity and peak areas by improving ionization and reducing TFA adduct formation.
Graphical abstract
•Development of Mucin based affinity chromatography stationary phases.•Study of the influence of the immobilization chemistry on retention.•Investigation of the influence of mobile phase composition ...on the retention process by experimental design.•Screening of 41 pharmaceutically active ingredients for their interaction with mucin.
Mucin glycoproteins belong to a class of high molecular weight, heavy glycosylated, proteins that together with water, salts and lipids constitute mucous secretions. Particular disease states (e.g. obstructive chronic bronchitis and ovarian tumor) are known to modify the composition and the thickness of those barriers. Therefore, it is important to address whether the absorption of potential drug candidates to be administered is influenced by the presence of interaction with this class of proteins. Typically, the methods adopted to characterize drug–protein interaction are dialysis, ultrafiltration and gel filtration. Besides these, bio-affinity chromatographic methods have demonstrated to be valuable tools offering the advantageous characteristics such as simplicity, efficiency, high-throughput capability and robustness. The present contribution reports on the synthesis and analytical characterization of a new chromatographic stationary phase based on covalently immobilized mucin and explores the use of LC-UV affinity zonal chromatography as a tool to screen drugs for their affinity to mucin. A series of different binding chemistries for the covalent linkage of mucin to silica-based supports as well as distinct immobilization protocols (static and dynamic) have been evaluated in order to optimize surface coverage. Resultant stationary phases have been characterized chromatographically by studying the effect of mobile phase and analyte structure on the distribution and retention of test compounds. As conclusive study, we report the evaluation of the retention characteristics of 41 drug-like compounds (having heterogeneous chemical properties) for their interaction with this novel stationary phase.
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