RESUMO
Cross-linkers play a critical role in capturing protein dynamics in chemical cross-linking mass spectrometry techniques. Various types of cross-linkers with different backbone features are widely used in the study of proteins. However, it is still not clear how the cross-linkers' backbone affect their own structure and their interactions with proteins. In this study, we systematically characterized and compared methylene backbone and polyethylene glycol (PEG) backbone cross-linkers in terms of capturing protein structure and dynamics. The results indicate the cross-linker with PEG backbone have a better ability to capture the inter-domain dynamics of calmodulin, adenylate kinase, maltodextrin binding protein and dual-specificity protein phosphatase. We further conducted quantum chemical calculations and all-atom molecular dynamics simulations to analyze thermodynamic and kinetic properties of PEG backbone and methylene backbone cross-linkers. Solution nuclear magnetic resonance was employed to validate the interaction interface between proteins and cross-linkers. Our findings suggest that the polarity distribution of PEG backbone enhances the accessibility of the cross-linker to the protein surface, facilitating the capture of sites located in dynamic regions. By comprehensively benchmarking with disuccinimidyl suberate (DSS)/bis-sulfosuccinimidyl-suberate(BS3), bis-succinimidyl-(PEG)2 revealed superior advantages in protein dynamic conformation analysis in vitro and in vivo, enabling the capture of a greater number of cross-linking sites and better modeling of protein dynamics. Furthermore, our study provides valuable guidance for the development and application of PEG backbone cross-linkers.
Assuntos
Polietilenoglicóis , Proteínas , Polietilenoglicóis/química , Proteínas/química , Espectrometria de Massas , Conformação Proteica , Simulação de Dinâmica MolecularRESUMO
In this work, a micron-sized flower-like metal-organic frameworks (MOFs)-based boronate-affinity sandwich-type immunoassay was fabricated for the dual-mode glycoprotein assay. For proof of concept, the flower-like MOFs were synthesized from transition Cu nodes and tetrakis (4-carboxyphenyl) porphyrin (TCPP) ligands by spontaneous standing assembly. In addition, the specificity toward glycoprotein involved the antigen recognition as well as covalent bonding via the boronate-glycan affinity, and the immediate signal responses were initiated by textural decomposition of the flower-like MOFs. Intriguingly, Cu nodes, of which the valence state is dominant by CuI species, can endow the Fenton-like catalytic reaction of the fluorogenic substrate for generating fluorescence signals. For benefits, TCPP ligands, in which each TCPP molecule has four guest donors, can provide multiple valences for the assembly of cyclodextrin-capped gold nanoparticles via host-guest interaction for colorimetry output. Albeit important, the scaling micrometer patterns for the flower-like MOFs carrying numerous Cu nodes and TCPP ligands can also function as amplifying units, signifying the output signal. The detection limit of the dual-mode glycoprotein assay can reach 10.5 nM for the fluorescence mode and 18.7 nM for the colorimetry mode, respectively. Furthermore, the merits of harvesting different signal generators toward the multimodal readout patterns can allow the mutual verification and make the analytical results more reliable. Collectively, our proposed assay may offer a new idea in combining the inherent textural merits from MOFs for dual signal generators, which can also emphasize accurate detection capability for glycoprotein assay.
Assuntos
Glicoproteínas , Estruturas Metalorgânicas , Estruturas Metalorgânicas/química , Glicoproteínas/análise , Glicoproteínas/química , Cobre/química , Porfirinas/química , Imunoensaio/métodos , Ouro/química , Nanopartículas Metálicas/química , Humanos , Ácidos Borônicos/química , Limite de Detecção , Tamanho da PartículaRESUMO
Small extracellular vesicles (sEVs) are proven to hold great promise for diverse therapeutic and diagnostic applications. However, batch preparation of sEVs with high purity and bioactivity is a prerequisite for their clinical translations. Herein, we present an electric field assisted tangential flow filtration system (E-TFF), which integrates size-based filtration with electrophoretic migration-based separation to synergistically achieve the isolation of high-quality sEVs from cell culture medium. Compared with the gold-standard ultracentrifugation (UC) method, E-TFF not only improved the purity of sEVs by 1.4 times but also increased the yield of sEVs by 15.8 times. Additionally, the entire isolation process of E-TFF was completed within 1 h, about one-fourth of the time taken by UC. Furthermore, the biological activity of sEVs isolated by E-TFF was verified by co-incubation of sEVs derived from human umbilical cord mesenchymal stem cells (hUCMSCs) with HT22 mouse hippocampal neuronal cells exposed to amyloid-ß (Aß). The results demonstrated that the sEVs isolated by E-TFF exhibited a significant neuroprotective effect. Overall, the E-TFF platform provides a promising and robust strategy for batch preparation of high-quality sEVs, opening up a broad range of opportunities for cell-free therapy and precision medicine.
RESUMO
Marine microbes drive pivotal transformations in planetary-scale elemental cycles and have crucial impacts on global biogeochemical processes. Metaproteomics is a powerful tool for assessing the metabolic diversity and function of marine microbes. However, hundreds of liters of seawater are required for normal metaproteomic analysis due to the sparsity of microbial populations in seawater, which poses a substantial challenge to the widespread application of marine metaproteomics, particularly for deep seawater. Herein, a sensitive marine metaproteomics workflow, named sensitive marine metaproteome analysis (SMMP), was developed by integrating polycarbonate filter-assisted microbial enrichment, solid-phase alkylation-based anti-interference sample preparation, and narrow-bore nanoLC column for trace peptide separation and characterization. The method provided more than 8500 proteins from 1 L of bathypelagic seawater samples, which covered diverse microorganisms and crucial functions, e.g., the detection of key enzymes associated with the Wood-Ljungdahl pathway. Then, we applied SMMP to investigate vertical variations in the metabolic expression patterns of marine microorganisms from the euphotic zone to the bathypelagic zone. Methane oxidation and carbon monoxide (CO) oxidation were active processes, especially in the bathypelagic zone, which provided a remarkable energy supply for the growth and proliferation of heterotrophic microorganisms. In addition, marker protein profiles detected related to ammonia transport, ammonia oxidation, and carbon fixation highlighted that Thaumarchaeota played a critical role in primary production based on the coupled carbon-nitrogen process, contributing to the storage of carbon and nitrogen in the bathypelagic regions. SMMP has low microbial input requirements and yields in-depth metaproteome analysis, making it a prospective approach for comprehensive marine metaproteomic investigations.
Assuntos
Proteômica , Água do Mar , Água do Mar/microbiologia , Água do Mar/química , Proteômica/métodos , Microbiota , Proteoma/análise , Proteoma/metabolismo , Metano/metabolismo , Metano/análise , Bactérias/metabolismo , Bactérias/isolamento & purificação , Oxirredução , Monóxido de Carbono/análise , Monóxido de Carbono/metabolismoRESUMO
Nonspecific cross-linker can provide distance restraints between surface residues of any type, which could be used to investigate protein structure construction and protein-protein interaction (PPI). However, the vast number of potential combinations of cross-linked residues or sites obtained with such a cross-linker makes the data challenging to analyze, especially for the proteome-wide applications. Here, we developed SpotLink software for identifying site nonspecific cross-links at the proteome scale. Contributed by the dual pointer dynamic pruning algorithm and the quality control of cross-linking sites, SpotLink identified > 3000 cross-links from human cell samples within a short period of days. We demonstrated that SpotLink outperformed other approaches in terms of sensitivity and precision on the datasets of the simulated succinimidyl 4,4'-azipentanoate dataset and the condensin complexes with known structures. In addition, some valuable PPI were discovered in the datasets of the condensin complexes and the HeLa dataset, indicating the unique identification advantages of site nonspecific cross-linking. These findings reinforce the importance of SpotLink as a fundamental characteristic of site nonspecific cross-linking technologies.
Assuntos
Proteoma , Software , Algoritmos , Reagentes de Ligações Cruzadas/química , HumanosRESUMO
MOTIVATION: Chemical cross-linking combined with mass spectrometry (CXMS) is now a well-established method for profiling existing protein-protein interactions (PPIs) with partially known structures. It is expected to map the results of CXMS with existing structure databases to study the protein dynamic profile in the structure analysis. However, currently available structure-based analysis software suffers from the difficulty of achieving large-scale analysis. Besides, it is infeasible for structure analysis and data mining on a large scale, since of lacking global measurement of dynamic structure mapping results. RESULTS: ComMap (protein complex structure mapping) is a software designed to perform large-scale structure-based mapping by integrating CXMS data with existing structures. It allows complete the distance calculation of PPIs with existing structures in batch within minutes and provides scores for different PPI-structure pairs of testable hypothetical structural dynamism via a global view. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Proteínas , Software , Reagentes de Ligações Cruzadas/química , Proteínas/química , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodosRESUMO
Neurotransmitters (NTs) and neuromodulators (NMs) are two of the most important neurochemicals in the brain, and their imbalances in specific brain regions are thought to underlie certain neurological disorders. We present an on-tissue chemoselective derivatization mass spectrometry imaging (OTCD-MSI) method for the simultaneous mapping of NTs and NMs. Our derivatization system consists of a pyridiniumyl-benzylboronic acid based derivatization reagent and pyrylium salt, which facilitate covalent charge labeling of molecules containing cis-diol and primary amino, respectively. These derivatization systems improved the detection sensitivity of matrix-assisted laser desorption/ionization (MALDI)-MSI and simplified the identification of amino NTs and nucleoside NMs by the innate chemoselectivity of derivatization reagents and the unique isotopic pattern of boron-derivative reagents. We demonstrated the ability of the developed method on brain sections from a hypoxia mouse model and control. The simultaneous imaging of NTs and NMs provided a method for exploring how hypoxic stress and drugs affect specific brain regions through neurotransmitter modulation.
Assuntos
Encéfalo , Nucleosídeos , Camundongos , Animais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neurotransmissores/análise , Modelos Animais de DoençasRESUMO
Various physiological activities and metabolic reactions of cells need to be carried out under the corresponding pH environment. Intracellular GSH as an acid tripeptide and an important reducing substance also plays an important role in maintaining cellular acid-base balance and redox balance. Therefore, developing a method to monitor pH and GSH and their changes in cells is necessary. Herein, we developed a novel turn-on fluorescent silicon nanoparticles (SiNPs) using N-(2-aminoethyl)-3-aminopropyltrimethoxysilane as the silicon source and dithiothreitol as the reducing agent via a one-pot hydrothermal method. It was worth mentioning that the fluorescence intensity of the SiNPs increased along with the acidity increase, making the SiNPs have excellent pH and GSH sensing capability. Furthermore, the pH and GSH sensing performance of the SiNPs in the cell was verified by confocal imaging and flow cytometry experiment. Based on the above, the prepared SiNPs had the potential to be used as an intracellular pH and GSH multimode fluorescent sensing platform and exhibited the ability to distinguish between normal cells and cancer cells.
Assuntos
Nanopartículas , Silício , Silício/química , Nanopartículas/química , Corantes Fluorescentes/química , Concentração de Íons de HidrogênioRESUMO
Comprehensive characterization of membrane proteins at the level of proteoforms in complex biological samples by top-down mass spectrometry (MS) is of vital importance in revealing their precise functions. However, severe peak broadening in the separation of hydrophobic membrane proteins, caused by resistance to mass transfer and strong adsorption on separation materials, leads to MS spectra overlap and signal suppression, which makes against the in-depth research on membrane proteoforms. Herein, C8-functional amine-bridged hybrid monoliths with an interconnected macroporous structure were developed by the in situ one-step sol-gel reaction of triethoxy(octyl)silane and bis[3-(trimethoxysilyl)propyl]amine in capillaries. Due to the unique macroporous structure and bridged secondary amino groups in the framework, the monolith possessed reduced resistance to mass transfer, low nonspecific adsorption, and electrostatic repulsion to membrane proteins. These features tremendously alleviated peak broadening in the separation of membrane proteins, thus outperforming traditional reversed-phase columns in top-down characterization of membrane proteoforms. With this monolith, a total of 3100 membrane proteoforms were identified in the mouse hippocampus, representing the largest membrane proteoform database obtained by top-down analysis so far. The identified membrane proteoforms revealed abundant information, including combinatorial post-translational modifications (PTMs), truncation, and transmembrane domains. Furthermore, the proteoform information was integrated into the interaction network of membrane protein complexes involved in oxidative phosphorylation processing, opening up new opportunities to uncover more detailed molecular basis and interaction in the biological processes.
Assuntos
Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem , Animais , Camundongos , Espectrometria de Massas em Tandem/métodos , Proteínas de MembranaRESUMO
The outbreak of coronavirus disease 2019 (COVID-19) has overwhelmed the global economy and human well-being. On account of the sharp increase in test demand, there is a need for an accurate and alternative diagnosis method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, with the aim to specifically identify the trace SARS-CoV-2 S1 glycoprotein, we developed a high-sensitivity and high-selectivity diagnostic method based on the targeted parallel reaction monitoring (PRM) assay of eight selected peptides. This study emphasizes the outstanding detection sensitivity of 0.01 pg of the SARS-CoV-2 S1 glycoprotein even in the interference of other structural proteins, which to our knowledge is the current minimum limit of detection for the SARS-CoV-2 S1 glycoprotein. This technology could further identify 0.01 pg of the SARS-CoV-2 S1 glycoprotein in a spike pseudovirus, revealing its practical effectiveness. All our preliminary results throw light on the capability of the mass spectrometry-based targeted PRM assay to identify SARS-CoV-2 as a practicable orthogonal diagnostic tool. Furthermore, this technology could be extended to other pathogens (e.g., MERS-CoV S1 protein or SARS-CoV S1 protein) by quickly adjusting the targeted peptides of MS data acquisition. In summary, this strategy is universal and flexible and could be quickly adjusted to detect and discriminate different mutants and pathogens.
Assuntos
COVID-19 , Coronavírus da Síndrome Respiratória do Oriente Médio , Humanos , SARS-CoV-2 , Glicoproteínas , Espectrometria de MassasRESUMO
Photosynthesis, as the core of solar energy biotransformation, is driven by photosynthetic membrane protein complexes in plants and algae. Current methods for intracellular photosynthetic membrane protein complex analysis mostly require the separation of specific chloroplasts or the change of the intracellular environment, which causes the missing of real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membrane protein complexes in the chloroplasts of living Chlamydomonas reinhardtii (C. reinhardtii) cells under cultural conditions. Poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles were fabricated to deliver bis(succinimidyl)propargyl with a nitro compound (BSPNO) into the chloroplasts to crosslink photosynthetic membrane protein complexes. After the in vivo crosslinked protein complexes were extracted and digested, mass spectrometry was employed to detect lysine-specific crosslinked peptides for further elucidating the protein conformations and interactions. With this method, the weak interactions between extrinsic proteins in the luminal side (PsbL and PsbH) and the core subunits (CP47 and CP43) in photosynthetic protein complexes were directly captured in living cells. Additionally, the previously uncharacterized protein (Cre07.g335700) was bound to the light-harvesting proteins, which was related to the biosynthesis of light-harvesting antennae. These results indicated that in vivo analysis of photosynthetic protein complexes based on crosslinker nanocarriers was expected to not only figure out the difficulty in the study of photosynthetic protein complexes in living cells but also provide an approach to explore transient and weak interactions and the function of uncharacterized proteins.
Assuntos
Chlamydomonas reinhardtii , Complexo de Proteínas do Centro de Reação Fotossintética , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Membrana/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Complexos de Proteínas Captadores de Luz/metabolismo , CloroplastosRESUMO
Conventional cyanine dyes exist as "always-on" fluorescent probes leading to inevitable background signals which often limit their performance and scope of applications. To develop specific fluorescent probes with high sensitivity and robust OFF/ON switching for targeting G4s, we introduced aromatic heterocycles through conjugation with polymethine chains to construct a rotor-π system. Here, a universal strategy is presented to synthesize pentamethine cyanines with different aromatic heterocycle substituents on the meso-polymethine chain. In these probes, SN-Cy5-S is self-quenched in aqueous solution due to H-aggregation. The structure indicates that SN-Cy5-S with a flexible meso-benzothiophenyl rotor conjugated to the cyanine backbone matches adaptively with G-tetrad planes, enhancing π-π stacking and resulting in triggered fluorescence. This allows recognition of G-quadruplexes due to the synergy of disaggregation-induced emission (DIE) and inhibited twisted intramolecular charge-transfer effects. This combination leads to a robust lighting-up fluorescence response for c-myc G4 with superior fluorescence enhancement (98-fold), allowing for a low detection limit of 1.51 nM, which is much more sensitive than the previously reported DIE-based G4 probes (22-83.5 nM). In addition, the superior imaging properties and rapid internalization time (5 min) in mitochondria allow SN-Cy5-S to also have a high potential for mitochondrially targeting anti-cancer therapy.
Assuntos
Corantes Fluorescentes , Quadruplex G , Corantes Fluorescentes/química , Iluminação , MitocôndriasRESUMO
Chemical cross-linking coupled with mass spectrometry (XL-MS) is an important technique for the structural analysis of protein complexes where the coverage of amino acids and the identification of cross-linked sites are crucial. Photo-cross-linking has multisite reactivity and is valuable for the structural analysis of chemical cross-linking. However, a high degree of heterogeneity results from this multisite reactivity, which results in samples with higher complexity and lower abundance. Additionally, the applicability of photo-cross-linking is limited to purified protein complexes. In this work, we demonstrate a photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with the reactive groups of N-hydroxysuccinimide ester and diazirine, as well as the click-enrichable alkyne group. Photo-cross-linkers can provide higher site reactivity for proteins that contain only a small number of lysine residues, thereby complementing the more commonly used lysine-targeting cross-linkers. By systematically analyzing proteins with differing lysine contents and differing flexibilities, we demonstrated clear enhancement in structure elucidation for proteins containing less lysine and with high flexibility. In addition, enrichment approaches of alkynyl-azide click chemistry conjugated with biotin-streptavidin purification (coinciding with parallel orthogonal digestion) improved the identification coverage of cross-links. We show that this photo-cross-linking approach can be used for membrane proteome-wide complex analysis. This method led to the identification of a total of 14066 lysine-X cross-linked site pairs from a total of 2784 proteins. Thus, this cross-linker is a valuable addition to a photo-cross-linking toolkit and improves the identification coverage of XL-MS in functional structure analysis.
Assuntos
Diazometano , Lisina , Lisina/química , Aminoácidos/química , Espectrometria de Massas/métodos , Proteoma , Reagentes de Ligações Cruzadas/químicaRESUMO
Protein dynamics play a crucial role in their diverse functions. The intracellular environment significantly influences protein dynamics, particularly for intrinsically disordered proteins (IDPs). To comprehensively capture structural information from various proteins within cells and characterize protein dynamics, chemical cross-linking mass spectrometry was employed. In this study, we introduce a hierarchical decoding strategy that enables the investigation of protein dynamics in vivo. Computational analysis based on distance restraints derived from cross-links is used to infer protein dynamics in cells. To facilitate this analysis, we leverage the prior structure obtained from AlphaFold2. By employing this strategy, we can characterize the full-length structure of multi-domain proteins taking into account their distinct dynamic features. Furthermore, by combining restraint sampling with an unbiased sampling and evaluation approach, we can provide a comprehensive description of the intrinsic motion of IDPs. Consequently, the hierarchical strategy we propose holds significant potential in advancing our understanding of the molecular mechanisms that undelie protein functions in cells.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Espectrometria de Massas , Conformação Proteica , Simulação de Dinâmica MolecularRESUMO
Chemical cross-linking mass spectrometry (CXMS) has emerged as a powerful technology to analyze protein complexes. However, the progress of in vivo CXMS studies has been limited by cross-linking biocompatibility and data analysis. Herein, a glycosidic bond-based MS-cleavable cross-linker of trehalose disuccinimidyl ester (TDS) was designed and synthesized, which was fragmented in MS under CID/HCD to simplify the cross-linked peptides into conventional single peptides via selective cleavage between glycosidic and peptide bonds under individual MS collision energy. Consequently, the cross-linking identification accuracy and throughput were significantly enhanced, and the popular MS mode of stepped HCD was allowed. In addition, TDS showed proper cell-penetrating properties while being highly water-soluble, making it non-DMSO dependent during solubilization. Collectively, TDS provides a promising toolkit for CXMS characterization of living systems with high biocompatibility and accuracy.
Assuntos
Glicosídeos , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Peptídeos/química , Reagentes de Ligações Cruzadas/químicaRESUMO
Top-down proteomics is challenged by the high complexity of biological samples. The coelution of intact proteins results in overlapped mass spectra, and hence, an increased peak capacity for protein separation is needed. Herein, ethane-bridged hybrid monoliths with well-defined large mesopores were successfully prepared based on the sol-gel condensation of 1,2-bis(trimethoxysilyl)ethane and tetramethoxysilane, followed by two-step base etching of the Si-O-Si domain while maintaining the Si-C-C-Si domain in the structure. Relatively homogeneous macropores of 1.1 µm and large mesopores of 24 nm were obtained, permitting fast mass transfer of large molecules and efficient diffusion without obstruction. The use of less hydrophobic C1 ligand further sharpened the peak shape and improved peak capacity. A 120 cm-long capillary column was used for top-down proteomic analysis of E. coli lysates under low backpressure with 16 MPa. High peak capacity of 646 was achieved within 240 min gradient. With MS/MS analysis, 959 proteoforms corresponding to 263 proteins could be unambiguously identified from E. coli lysates in a single run. Furthermore, to illustrate the separation performance for large proteoforms, such monoliths were applied to top-down analysis of the SEC fraction of E. coli lysates with Mw ranging from 30 to 70 kDa. With highly effective separation, 347 large proteoforms with Mw higher than 30 kDa were detected in the single 75 min run. These results showed great potential for top-down proteomic analysis in complex samples.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Escherichia coli/química , Etano/química , Proteínas/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
For proteomic analysis based on mass spectrometry (MS), high-performance peptide separation under MS-friendly conditions is of importance. To this end, a novel kind of amine-bridged hybrid monolith was developed by the sol-gel reaction of bis[3-(trimethoxysilyl)propyl]amine and allyltrimethoxysilane, followed by "thiol-ene" click functionalization of C18 groups. With the secondary amino groups bridged in the framework, the nonspecific adsorption from silanol groups could be decreased, so that peptide peak tailing under MS-friendly conditions was reduced, and half peak width was narrowed. Furthermore, such materials were facilely in situ prepared in the very narrow bore capillary with low backpressure for proteomic analysis of limited amounts of samples. Finally, 16,692 unique peptides corresponding to 3698 protein groups could be averagely identified from 10 ng Hela cell digests in a single 65 min run, and 5257 peptides corresponding to 1062 protein groups could be averagely identified from 200 pg digests in a single 60 min run. Such high sensitivity could be attributed to the decreased nonspecific adsorption, the narrowed peak width, and the miniaturization of the column. It is shown that such monoliths are promising for highly sensitive proteomic analysis, including single-cell proteomics.
Assuntos
Aminas , Proteômica , Células HeLa , Humanos , Espectrometria de Massas , Peptídeos/química , Proteômica/métodosRESUMO
For bottom-up proteomics, peptide separation with high peak capacity under MS-compatible conditions is of vital significance to increase proteome coverage. Herein, a surface-charged ethane-bridged hybrid monolithic column was prepared based on the efficient ring-opening reaction of N-methyl-aza-2,2,4-trimethyl-silacyclopentane after C18-functionalization. The existence of secondary amino groups on the surface was beneficial to reduce the secondary interactions of silanol groups and increase peak capacity for peptide separation with MS-compatible mobile phases (e.g., using 0.1% FA as the mobile phase modifier). Such columns offered a 4-fold increase in peak capacity compared with ethane-bridged hybrid monolithic columns without surface charge modification. By a 100 cm length surface-charged ethane-bridged hybrid capillary column, high peak capacity of 700 was achieved within a 240 min gradient for the separation of Hela tryptic peptides with 0.1% FA-containing mobile phases, under the low backpressure of â¼200 bar. On average, 44493 ± 459 peptides corresponding to 5148 ± 47 proteins were identified from 750 ng Hela tryptic digests. Finally, the surface-charged ethane-bridged hybrid monolithic column was successfully applied in the quantitative proteomic analysis of dopaminergic neuron death model of N-methyl-4-phenylpyridinium iodide induced SH-SY5Y cells. These results demonstrated great promise of such surface-charged ethane-bridged hybrid monolithic columns for bottom-up proteomic analysis in complex samples.
Assuntos
Neuroblastoma , Proteômica , Etano , Humanos , Peptídeos/análise , Proteoma , Proteômica/métodosRESUMO
The coverage of chemical crosslinking coupled with mass spectrometry (CXMS) is of great importance to determine its ability for deciphering protein structures. At present, N-hydroxysuccinimidyl (NHS) ester-based crosslinkers targeting lysines have been predominantly used in CXMS. However, they are not always effective for some proteins with few lysines. Other amino acid residues such as carboxyl could be crosslinked to complement lysines and improve the crosslinking coverage of CXMS, but the low intrinsic chemical reactivity of carboxyl compromises the application of carboxyl-selective crosslinkers for complex samples. To enhance the crosslinking efficiency targeting acidic residues and realize in-depth crosslinking analysis of complex samples, we developed three new alkynyl-enrichable carboxyl-selective crosslinkers with different reactive groups such as hydrazide, amino, and aminooxy. The crosslinking efficiencies of the three crosslinkers were systematically evaluated, giving the best reactivity of the amino-functionalized crosslinker BAP. Furthermore, BAP was extended to the crosslinking analysis of Escherichia coli lysate in combination with efficient crosslink enrichment. A total of 1291 D/E-D/E crosslinks involved in 392 proteins were identified under a false discovery rate (FDR) of ≤1%. Obvious structural complementarity of BAP was exhibited to the lysine-targeting crosslinker, facilitating the capability of CXMS for protein structure elucidation. To the best of our knowledge, this was the first time for the carboxyl-selective crosslinker to achieve proteome-wide crosslinking analysis of the whole cell lysate. Collectively, we believe that this work not only expands on a promising toolkit of CXMS targeting acidic residues but also provides a valuable guideline to advance the performance of carboxyl-selective crosslinkers.
Assuntos
Aminoácidos , Proteínas , Reagentes de Ligações Cruzadas/química , Lisina , Espectrometria de Massas/métodos , Proteínas/químicaRESUMO
Accurate proteome quantitation is of great significance to deeply understand various cellular and physiological processes. Since a1 ions, generated from dimethyl-labeled peptides, exhibited high formation efficiency (up to 99%) and enhanced intensities (2.34-fold by average) in tandem mass spectra, herein, we proposed an a1 ion-based proteome quantitation (APQ) method, which showed high quantitation accuracy (relative errors < 7%) and precision (median coefficients of variation ≤ 11%) even in a 20-fold dynamic range. Notably, due to the mass differences of a1 ions from peptides with different N-terminal amino acids, APQ demonstrated interference-free capacity by distinguishing target peptides from the coisolated ones. By designing an isobaric dimethyl labeling strategy, we achieved simultaneous proteome-wide measurements across up to eight samples. Using APQ to quantify the time-resolved proteomic profiles during a TGF-ß-induced epithelial-mesenchymal transition, we found many differentially expressed proteins associated with fatty acid degradation, indicating that fatty acid metabolism reprogramming occurred during the process. The APQ method combines high quantitation accuracy with multiplexing capacity, which is suitable for deep mining and understanding of dynamic biological processes.