ABSTRACT
Recently, alcohol-based draw solute (DS), i.e., alcohol with water, is one of the trending research topics in forward osmosis (FO) because of its performance and ease of regeneration. Nevertheless, the higher reverse solute flux (RSF) of the alcohol-based DS hinders its commercialization in water and wastewater treatment applications. This research aims to minimize the RSF of the alcohol-based DS in FO by investigating the possibility of using alcohol-alcohol-based draw solutes for the first time. Three alcohol-alcohol-based draw solutions, namely, (1) E70 + IPA30 (ethanol: 70% + isopropanol: 30%), (2) E40 + IPA60 (ethanol: 40% + isopropanol: 60%), and (3) E10 + IPA90 (ethanol: 10% + isopropanol: 90%), were prepared and the properties (including osmolality, shear stress, and viscosity) of the DS were first investigated followed by the parametric investigation (concerning temperature and concentration). The results were further analyzed with the fixed-point iterative method in MATLAB to obtain the performance parameters. Results reveal that the E10 + IPA90 mixture yields a lower RSF of 40.62 g/m2/h and specific reverse solute flux of 3.78 g/L with a considerably good water flux and recovery percentage of 11.47 LMH and 26.29%, respectively, as compared to other DS E70 + IPA30 and E40 + IPA60 at 25 °C. Thus, E10 + IPA90 is recommended as a potential candidate to be used as a DS in FO.
Subject(s)
Osmosis , Water Purification , Water Purification/methods , Alcohols/chemistry , Wastewater/chemistryABSTRACT
ConspectusOne-dimensional organic nanotubes feature unique properties, such as confined chemical environments and transport channels, which are highly desirable for many applications. Advances in synthetic methods have enabled the creation of different types of organic nanotubes, including supramolecular, hydrogen-bonded, and carbon nanotube analogues. However, challenges associated with chemical and mechanical stability along with difficulties in controlling aspect ratios remain a significant bottleneck. The fascination with structured porous materials has paved the way for the emergence of reticular solids such as metal-organic frameworks (MOFs), covalent organic frameworks (COFs), and organic cages. Reticular materials with tubular morphology promise architectural stability with the additional benefit of permeant porosity. Despite this, the current synthetic approaches to these reticular nanotubes focus more on structural design resulting in less reliable morphological uniformity. This Account, highlights the design motivation behind various classes of organic nanotubes, emphasizing their porous interior space. We explore the strategic assembly of organic nanotubes based on their bonding characteristics, from weak supramolecular to robust covalent interactions. Special attention is given to reticular nanotubes, which have gained prominence over the past two decades due to their distinctive micro and mesoporous structures. We examine the synergy of covalent and noncovalent interactions in constructing assembly of these nanotube structures.This Account furnishes a comprehensive overview of our efforts and advancements in developing porous covalent organic nanotubes (CONTs). We describe a general synthetic approach for creating robust imine-linked nanotubes based on the reticular chemistry principles. The use of spatially oriented tetratopic triptycene-based amine and linear ditopic aldehyde building blocks facilitates one-dimensional nanotube growth. The interplay between directional covalent bonds and solvophobic interactions is crucial for forming uniform, well-defined, and high aspect ratio nanotubes. The nanotubes derive their permeant porosity and thermal and chemical stability from their covalent architecture. We also highlight the adaptability of our synthetic methodology to guide the transformation of one-dimensional nanotubes to toroidal superstructures and two-dimensional thin fabrics. Such morphological transformation can be directed by tuning the reaction time or incorporating additional intermolecular interactions to control the intertwining behavior of individual nanotubes. The cohesion of covalent and noncovalent interactions in the tubular nanostructures manifests superior viscoelastic mechanical properties in the assembled CONT fabrics. We establish a strong correlation between structural framework design and nanostructures by translating reticular synthesis to morphological space and gaining insights into the assembly processes. We anticipate that the present Account will lay the foundation for exploring new designs and chemistry of organic nanotubes for many application platforms.
ABSTRACT
Alzheimer's disease (AD) is the most prevalent form of dementia, disproportionately affecting women in disease prevalence and progression. Comprehensive analysis of the serum proteome in a common AD mouse model offers potential in identifying possible AD pathology- and gender-associated biomarkers. Here, we introduce a multiplexed, nondepleted mouse serum proteome profiling via tandem mass-tag (TMTpro) labeling. The labeled sample was separated into 475 fractions using basic reversed-phase liquid chromatography (RPLC), which were categorized into low-, medium-, and high-concentration fractions for concatenation. This concentration-dependent concatenation strategy resulted in 128 fractions for acidic RPLC-tandem mass spectrometry (MS/MS) analysis, collecting â¼5 million MS/MS scans and identifying 3972 unique proteins (3413 genes) that cover a dynamic range spanning at least 6 orders of magnitude. The differential expression analysis between wild type and the commonly used AD model (5xFAD) mice exhibited minimal significant protein alterations. However, we detected 60 statistically significant (FDR < 0.05), sex-specific proteins, including complement components, serpins, carboxylesterases, major urinary proteins, cysteine-rich secretory protein 1, pregnancy-associated murine protein 1, prolactin, amyloid P component, epidermal growth factor receptor, fibrinogen-like protein 1, and hepcidin. The results suggest that our platform possesses the sensitivity and reproducibility required to detect sex-specific differentially expressed proteins in mouse serum samples.
Subject(s)
Alzheimer Disease , Humans , Male , Mice , Female , Animals , Alzheimer Disease/metabolism , Tandem Mass Spectrometry/methods , Proteome/analysis , Reproducibility of Results , Chromatography, Reverse-PhaseABSTRACT
Covalent organic frameworks (COFs) are ideal host matrices for biomolecule immobilization and biocatalysis due to their high porosity, various functionalities, and structural robustness. However, the porosity of COFs is limited to the micropore dimension, which restricts the immobilization of enzymes with large volumes and obstructs substrate flow during enzyme catalysis. A hierarchical 3D nanostructure possessing micro-, meso-, and macroporosity could be a beneficial host matrix for such enzyme catalysis. In this study, we employed an in situ CO2 gas effervescence technique to induce disordered macropores in the ordered 2D COF nanostructure, synthesizing hierarchical TpAzo COF-foam. The resulting TpAzo foam matrix facilitates the immobilization of multiple enzymes with higher immobilization efficiency (approximately 1.5 to 4-fold) than the COF. The immobilized cellulolytic enzymes, namely ß-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (EG), remain active inside the TpAzo foam. The immobilized BGL exhibited activity in organic solvents and stability at room temperature (25 °C). The enzyme-immobilized TpAzo foam exhibited significant activity towards the hydrolysis of p-nitrophenyl-ß-d-glucopyranoside (BGL@TpAzo-foam: Km and Vmax = 23.5 ± 3.5 mM and 497.7 ± 28.0 µM min-1) and carboxymethylcellulose (CBH@TpAzo-foam: Km and Vmax = 18.3 ± 4.0 mg mL-1 and 85.2 ± 9.6 µM min-1 and EG@TpAzo-foam: Km and Vmax = 13.2 ± 2.0 mg mL-1 and 102.2 ± 7.1 µM min-1). Subsequently, the multi-enzyme immobilized TpAzo foams were utilized to perform a one-pot tandem conversion from carboxymethylcellulose (CMC) to glucose with high recyclability (10 cycles). This work opens up the possibility of synthesizing enzymes immobilized in TpAzo foam for tandem catalysis.
ABSTRACT
Peptide-based biomimetic catalysts are promising materials for efficient catalytic activity in various biochemical transformations. However, their lack of operational stability and fragile nature in non-aqueous media limit their practical applications. In this study, we have developed a cladding technique to stabilize biomimetic catalysts within porous covalent organic framework (COF) scaffolds. This methodology allows for the homogeneous distribution of peptide nanotubes inside the COF (TpAzo and TpDPP) backbone, creating strong noncovalent interactions that prevent leaching. We synthesized two different peptide-amphiphiles, C10FFVK and C10FFVR, with lysine (K) and arginine (R) at the C-termini, respectively, which formed nanotubular morphologies. The C10FFVK peptide-amphiphile nanotubes exhibit enzyme-like behavior and efficiently catalyze C-C bond cleavage in a buffer medium (pH 7.5). We produced nanotubular structures of TpAzo-C10FFVK and TpDPP-C10FFVK through COF cladding by using interfacial crystallization (IC). The peptide nanotubes encased in the COF catalyze C-C bond cleavage in a buffer medium as well as in different organic solvents (such as acetonitrile, acetone, and dichloromethane). The TpAzo-C10FFVK catalyst, being heterogeneous, is easily recoverable, enabling the reaction to be performed for multiple cycles. Additionally, the synthesis of TpAzo-C10FFVK thin films facilitates catalysis in flow. As control, we synthesized another peptide-amphiphile, C10FFVR, which also forms tubular assemblies. By depositing TpAzo COF crystallites on C10FFVR nanotubes through IC, we produced TpAzo-C10FFVR nanotubular structures that expectedly did not show catalysis, suggesting the critical role of the lysines in the TpAzo-C10FFVK.
ABSTRACT
Intrahepatic cholangiocarcinoma (iCCA) is characterized by its highly desmoplastic stroma. Myofibroblasts (MFs) are present both within the tumor mass (intratumoral MFs, iMFs) and at the tumor border (peritumoral MFs, pMFs). Using a spheroid-based coculture system, we show that the initial iCCA-pMF contact is growth suppressive to the tumor cells. However, prolonged iCCA-pMF interaction elicits significant tumor cell invasion and dissemination. We find that vascular cell adhesion molecule-1 (Vcam1) level is elevated in tumor cells in contact with pMFs but low in disseminated tumor cells both in vitro and in vivo. A gene regulatory network analysis of mouse and patient iCCA tumors and Vcam1 knockout (Vcam1KO) demonstrate a heavy involvement of Vcam1 in epithelial-to-mesenchymal transition. While Vcam1KO has only a limited impact on tumor cell growth in their monoculture, Vcam1KO spheroids exhibit instant dissemination and a severe growth defect when cocultured with pMFs. When transplanted into the liver, Vcam1KO iCCA cells show a similar increase in dissemination but a significant defect in establishing primary and metastatic tumors. Incomplete blocking of Vcam1 in vivo reduces the size but increase the number of metastatic lesions. Overall, our study shows a spatiotemporal regulation of iCCA growth and dissemination by pMFs in a Vcam1-dependent manner.
Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Humans , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolism , Myofibroblasts/metabolism , Cholangiocarcinoma/pathology , Bile Ducts, Intrahepatic/pathology , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/pathologyABSTRACT
The synthesis of homogeneous covalent organic framework (COF) thin films on a desired substrate with decent crystallinity, porosity, and uniform thickness has great potential for optoelectronic applications. We have used a solution-processable sphere transmutation process to synthesize 300 ± 20 nm uniform COF thin films on a 2 × 2 cm2 TiO2-coated fluorine-doped tin oxide (FTO) surface. This process controls the nucleation of COF crystallites and molecular morphology that helps the nanospheres to arrange periodically to form homogeneous COF thin films. We have synthesized four COF thin films (TpDPP, TpEtBt, TpTab, and TpTta) with different functional backbones. In a close agreement between the experiment and density functional theory, the TpEtBr COF film showed the lowest optical band gap (2.26 eV) and highest excited-state lifetime (8.52 ns) among all four COF films. Hence, the TpEtBr COF film can participate in efficient charge generation and separation. We constructed optoelectronic devices having a glass/FTO/TiO2/COF-film/Au architecture, which serves as a model system to study the optoelectronic charge transport properties of COF thin films under dark and illuminated conditions. Visible light with a calibrated intensity of 100 mW cm-2 was used for the excitation of COF thin films. All of the COF thin films exhibit significant photocurrent after illumination with visible light in comparison to the dark. Hence, all of the COF films behave as good photoactive substrates with minimal pinhole defects. The fabricated out-of-plane photodetector device based on the TpEtBr COF thin film exhibits high photocurrent density (2.65 ± 0.24 mA cm-2 at 0.5 V) and hole mobility (8.15 ± 0.64 ×10-3 cm2 V-1 S-1) compared to other as-synthesized films, indicating the best photoactive characteristics.
ABSTRACT
Schwannoma is a benign tumor originating from Schwann cells of the nerve sheath. Approximately 25-45% of the schwannomas are seen in the head and neck region and are found rarely in the oral cavity (only 1%). The most common intra-oral site is tongue, followed by floor of the mouth, buccal mucosa, palate, gingiva and lips. We report a rare case of schwannoma in the soft palate in a 22 years old female. She presented with 6 months history of a painless swelling in palate. The provisional diagnosis was made as some benign neoplasm of minor salivary gland. The tumour was excised intra-orally under general anesthesia. Histopathologic examination showed neural tissue arranged in predominantly Antoni A pattern and formation of verocay bodies. It is difficult to diagnose this tumor based on clinical appearance. Therefore final diagnosis can only be done after histopathological examination of the lesion. Prognosis is good and recurrence is unknown.
ABSTRACT
Translating the performance of covalent organic frameworks (COFs) from laboratory to macroscopic reality demands specific morphologies. Thus, the advancement in morphological modulation has recently gained some momentum. A clear understanding of nano- to macroscopic architecture is critical to determine, optimize, and improve performances of this atomically precise porous material. Along with their chemical compositions and molecular frameworks, the prospect of morphology in various applications should be discussed and highlighted. A thorough insight into morphology versus application will help produce better-engineered COFs for practical implications. 2D and 3D frameworks can be transformed into various solids such as nanospheres, thin films, membranes, monoliths, foams, etc., for numerous applications in adsorption, separation photocatalysis, the carbon dioxide reduction, supercapacitors, and fuel cells. However, the research on COF chemistry mainly focuses on correlating structure to property, structure to morphology, and structure to applications. Here, critical insights on various morphological evolution and associated applications are provided. In each case, the underlying role of morphology is unveiled. Toward the end, a correlation between morphology and application is provided for the future development of COFs.
ABSTRACT
Covalent organic frameworks (COFs) are promising hosts in heterogeneous catalysis. Herein, we report a dual metalation strategy in a single two-dimensional-COF TpBpy for performing a variety of C-N cross-coupling reactions. [Ir(ppy)2(CH3CN)2]PF6 [ppy = 2-phenylpyridine], containing two labile CH3CN groups, and NiCl2 are used as iridium and nickel-metal precursors, respectively, for postsynthetic decoration of the TpBpy COF. Moving from the traditional approach, we focus on the COF-backbone host for visible-light-mediated nickel-catalyzed C-N coupling reactions. The controlled metalation and recyclability without deactivation of both catalytic centers are unique with respect to previously reported coupling strategies. We performed various photoluminescence, electrochemical, kinetic, and Hammett correlation studies to understand the salient features of the catalyst and reaction mechanism. Furthermore, theoretical calculations delineated the feasibility of electron transfer from the Ir center to the Ni center inside the confined pore of the TpBpy COF. The dual metal anchoring within the COF backbone prevented nickel-black formation. The developed protocol enables selective and reproducible coupling of a diverse range of amines (aryl, heteroaryl, and alkyl), carbamides, and sulfonamides with electron-rich, neutral, and poor (hetero) aryl iodides up to 94% isolated yield. The reaction can also be performed on a gram scale. Furthermore, to establish the practical implementation of this approach, we have applied the synthetic strategy for the late-stage diversification of the derivatives of ibuprofen, naproxen, gemfibrozil, helional, and amino acids. The methodology could also be applied to synthesize pharmacophore N,5-diphenyloxazol-2-amine and Food and Drug Administration-approved drugs, including flufenamic acid, flibanserin, and tripelennamine.
Subject(s)
Metal-Organic Frameworks , Amines , Catalysis , Electrons , Light , Metal-Organic Frameworks/chemistry , Nickel/chemistryABSTRACT
Proteome profiling is a powerful tool in biological and biomedical studies, starting with samples at bulk, single-cell, or single-cell-type levels. Reliable methods for extracting specific cell-type proteomes are in need, especially for the cells (e.g., neurons) that cannot be readily isolated. Here, we present an innovative proximity labeling (PL) strategy for single-cell-type proteomics of mouse brain, in which TurboID (an engineered biotin ligase) is used to label almost all proteins in a specific cell type. This strategy bypasses the requirement of cell isolation and includes five major steps: (i) constructing recombinant adeno-associated viruses (AAVs) to express TurboID driven by cell-type-specific promoters, (ii) delivering the AAV to mouse brains by direct intravenous injection, (iii) enhancing PL labeling by biotin administration, (iv) purifying biotinylated proteins, followed by on-bead protein digestion, and (v) quantitative tandem-mass-tag (TMT) labeling. We first confirmed that TurboID can label a wide range of cellular proteins in human HEK293 cells and optimized the single-cell-type proteomic pipeline. To analyze specific brain cell types, we generated recombinant AAVs to coexpress TurboID and mCherry proteins, driven by neuron- or astrocyte-specific promoters and validated the expected cell expression by coimmunostaining of mCherry and cellular markers. Subsequent biotin purification and TMT analysis identified â¼10,000 unique proteins from a few micrograms of protein samples with excellent reproducibility. Comparative and statistical analyses indicated that these PL proteomes contain cell-type-specific cellular pathways. Although PL was originally developed for studying protein-protein interactions and subcellular proteomes, we extended it to efficiently tag the entire proteomes of specific cell types in the mouse brain using TurboID biotin ligase. This simple, effective in vivo approach should be broadly applicable to single-cell-type proteomics.
Subject(s)
Proteome , Proteomics , Animals , Biotinylation , Brain/metabolism , HEK293 Cells , Humans , Mice , Proteome/analysis , Proteomics/methods , Reproducibility of ResultsABSTRACT
Mass spectrometry (MS) has become a mainstream platform for comprehensive profiling of proteome, especially with the improvement of multiplexed tandem mass tag labeling coupled with two-dimensional liquid chromatography and tandem mass spectrometry (TMT-LC/LC-MS/MS). Recently, we have established a robust method for direct profiling of undepleted cerebrospinal fluid (CSF) proteome with the 16-plex TMTpro method, in which we optimized parameters in experimental steps of sample preparation, TMT labeling, LC/LC fractionation, tandem mass spectrometry, and computational data processing. The extensive LC fractionation not only enhances proteome coverage of the CSF but also alleviates ratio distortion of TMT quantification. The crucial quality control steps and improvements specific for the TMT16 analysis are highlighted. More than 3000 proteins can be quantified in a single experiment from 16 different CSF samples. This multiplexed method offers a powerful tool for profiling a variety of complex biofluids samples such as CSF, serum/plasma, and other clinical specimens.
Subject(s)
Proteome , Tandem Mass Spectrometry , Chromatography, Liquid , Gene Expression Profiling , ProteomicsABSTRACT
Empowered by crystalline ordered structures and homogeneous fabrication techniques, covalent organic frameworks (COFs) have been realized with uniform morphologies and isotropic properties. However, such homogeneity often hinders various surface-dependent properties observed in asymmetric nanostructures. The challenge remains to induce heterogeneity in COFs by creating an asymmetric superstructure such as a Janus thin film. In this regard, we propose a versatile yet straightforward interfacial layer-grafting strategy to fabricate free-standing Janus-type COF-graphene thin films. Herein, two-dimensional graphene sheets were utilized as the suitable grafter due to the possibility of noncovalent interactions between the layers. The versatility of the approach was demonstrated by fabricating two distinct Janus-type films, with the COF surface interwoven with nanofibers and nanospheres. The Janus-type films showcase opposing surface morphologies originating from graphene sheets and COF nanofibers or nanospheres, preserving the porosity (552-600 m2 g-1). The unique surface chemistries of the constituent layers further endow the films with orthogonal mechanical properties, as confirmed by the nanoindentation technique. Interestingly, the graphene sheets favor the Janus-type assembly of COF nanofibers over the nanospheres. This is reflected in the better nanomechanical properties of COFfiber-graphene films (Egraphene = 300-1200 MPa; ECOF = 15-60 MPa) compared to the COFsphere-graphene films (Egraphene = 11-14 MPa; ECOF = 2-5 MPa). These results indicate a direct relationship between the mechanical properties and homo/heterogeneity of Janus-type COF films.
ABSTRACT
Synthesis of covalent organic framework (COF) thin films on different supports with high crystallinity and porosity is crucial for their potential applications. We have designed a new synchronized methodology, residual crystallization (RC), to synthesize sub 10 nm COF thin films. These residual crystallized COF thin films showcase high surface area, crystallinity, and conductivity at room temperature. We have used interfacial crystallization (IC) as a rate-controlling tool for simultaneous residual crystallization. We have also diversified the methodology of residual crystallization by utilizing two different crystallization pathways: fiber-to-film (F-F) and sphere-to-film (S-F). In both cases, we could obtain continuous COF thin films with high crystallinity and porosity grown on various substrates (the highest surface area of a TpAzo COF thin film being 2093 m2 g-1). Precise control over the crystallization allows the synthesis of macroscopic defect-free sub 10 nm COF thin films with a minimum thickness of â¼1.8 nm. We have synthesized two COF thin films (TpAzo and TpDPP) using F-F and S-F pathways on different supports such as borosilicate glass, FTO, silicon, Cu, metal, and ITO. Also, we have investigated the mechanism of the growth of these thin films on various substrates with different wettability. Further, a hydrophilic support (glass) was used to grow the thin films in situ for four-probe system device fabrication. All residual crystallized COF thin films exhibit outstanding conductivity values. We could obtain a conductivity of 3.7 × 10-2 mS cm-1 for the TpAzo film synthesized by S-F residual crystallization.
ABSTRACT
Mass spectrometry-based proteomics empowers deep profiling of proteome and protein posttranslational modifications (PTMs) in Alzheimer's disease (AD). Here we review the advances and limitations in historic and recent AD proteomic research. Complementary to genetic mapping, proteomic studies not only validate canonical amyloid and tau pathways, but also uncover novel components in broad protein networks, such as RNA splicing, development, immunity, membrane transport, lipid metabolism, synaptic function, and mitochondrial activity. Meta-analysis of seven deep datasets reveals 2,698 differentially expressed (DE) proteins in the landscape of AD brain proteome (n = 12,017 proteins/genes), covering 35 reported AD genes and risk loci. The DE proteins contain cellular markers enriched in neurons, microglia, astrocytes, oligodendrocytes, and epithelial cells, supporting the involvement of diverse cell types in AD pathology. We discuss the hypothesized protective or detrimental roles of selected DE proteins, emphasizing top proteins in "amyloidome" (all biomolecules in amyloid plaques) and disease progression. Comprehensive PTM analysis represents another layer of molecular events in AD. In particular, tau PTMs are correlated with disease stages and indicate the heterogeneity of individual AD patients. Moreover, the unprecedented proteomic coverage of biofluids, such as cerebrospinal fluid and serum, procures novel putative AD biomarkers through meta-analysis. Thus, proteomics-driven systems biology presents a new frontier to link genotype, proteotype, and phenotype, accelerating the development of improved AD models and treatment strategies.
Subject(s)
Alzheimer Disease/metabolism , Nerve Tissue Proteins/metabolism , Proteome , Alzheimer Disease/etiology , Alzheimer Disease/genetics , Asymptomatic Diseases , Biomarkers , Blood Proteins/analysis , Cerebrospinal Fluid Proteins/analysis , Chromatography, Liquid , Cognitive Dysfunction/metabolism , Data Mining , Databases, Protein , Datasets as Topic , Humans , Meta-Analysis as Topic , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/genetics , Plaque, Amyloid/chemistry , Protein Processing, Post-Translational , Proteomics/methods , Tandem Mass SpectrometryABSTRACT
Nanomechanics signifies a key tool to interpret the macroscopic mechanical properties of a porous solid in the context of molecular-level structure. However, establishing such a correlation has proved to be significantly challenging in porous covalent organic frameworks (COFs). Structural defects or packing faults within the porous matrix, poor understanding of the crystalline assembly, and surface roughness are critical factors that contribute to this difficulty. In this regard, we have fabricated two distinct types of COF thin films by controlling the internal order and self-assembly of the same building blocks. Interestingly, the defect density and the nature of supramolecular interactions played a significant role in determining the corresponding thin films' stress-strain behavior. Thin films assembled from nanofibers (â¼1-2 µm) underwent large deformation on the application of small external stress (Tp-Azofiber film: E ≈ 1.46 GPa; H ≈ 23 MPa) due to weak internal forces. On the other hand, thin films threaded with nanospheres (â¼600 nm) exhibit a much stiffer and harder mechanical response (Tp-Azosphere film: E ≈ 15.3 GPa; H ≈ 66 MPa) due to strong covalent interactions and higher crystallinity. These porous COF films further exhibited a significant elastic recovery (â¼80%), ideal for applications dealing with shock-resistant materials. This work provides in-depth insight into the fabrication of industrially relevant crystalline porous thin films and membranes by addressing the previously unanswered questions about the mechanical constraints in COFs.
ABSTRACT
Tandem mass tag (TMT)-based mass spectrometry (MS) enables deep proteomic profiling of more than 10,000 proteins in complex biological samples but requires up to 100 µg protein in starting materials during a standard analysis. Here, we present a streamlined protocol to quantify more than 9000 proteins with 0.5 µg protein per sample by 16-plex TMT coupled with two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). In this protocol, we optimized multiple conditions to reduce sample loss, including processing each sample in a single tube to minimize surface adsorption, increasing digestion enzymes to shorten proteolysis and function as carriers, eliminating a desalting step between digestion and TMT labeling, and developing miniaturized basic pH LC for prefractionation. By profiling 16 identical human brain tissue samples of Alzheimer's disease (AD), vascular dementia (VaD), and non-dementia controls, we directly compared this new microgram-scale protocol to the standard-scale protocol, quantifying 9116 and 10,869 proteins, respectively. Importantly, bioinformatics analysis indicated that the microgram-scale protocol had adequate sensitivity and reproducibility to detect differentially expressed proteins in disease-related pathways. Thus, this newly developed protocol is of general application for deep proteomics analysis of biological and clinical samples at sub-microgram levels.