RESUMO
Extracellular vesicles (EVs) are cell-released vesicles that mediate intercellular communication by transferring bioactive cargo. Protein and RNA sorting into EVs has been extensively assessed, while selective enrichment of glycans in EVs remains less explored. In this study, a mass spectrometry-based approach, glycan node analysis (GNA), was applied to broadly assess the sorting of glycan features into EVs. Two metastatic variants (lung and bone) generated in mouse modes from the MDA-MB-231 human breast cancer cell line were assessed, as these EVs are known to contain distinct organotropic biomolecules. EVs were isolated from conditioned cell culture medium by tangential flow filtration and authenticated by standard techniques. GNA analysis revealed selective enrichment of several glycan features in EVs compared to the originating cells, particularly those associated with binding to the extracellular matrix, which was also observed in EVs from the parental MDA-MB-231 cell line (human pleural metastases). The bone-tropic variant displayed enrichment of distinct EV glycan features compared to the lung-tropic one. Additionally, the metastatic variants generated in mouse models displayed reduced EV glycan sorting compared to the parental metastatic cell line. This study represents the first comprehensive assessment of differences in glycan features between EVs and originating cells and provides evidence that the diversity of EV glycan sorting is reduced upon generation of variant cell lines in mouse models. Future research is likely to uncover novel mechanisms of EV glycan sorting, shed light on glycan features for EV authentication or biomarker purposes, and assess functional roles of the EV glycocode in (patho)physiology.
Assuntos
Neoplasias da Mama , Vesículas Extracelulares , Humanos , Animais , Camundongos , Feminino , Vesículas Extracelulares/metabolismo , Neoplasias da Mama/metabolismo , Biomarcadores/metabolismo , Proteínas/metabolismo , Polissacarídeos/análiseRESUMO
Glycan node analysis (GNA) is a molecularly bottom-up glycomics technique based on the relative quantification of glycan linkage-specific monosaccharide units ("glycan nodes"). It was originally applied to blood plasma/serum, where it detected and predicted progression, reoccurrence, and survival in different types of cancer. Here, we have adapted this technology to previously inaccessible membrane glycoproteins from cultured cells. The approach is facilitated by methanol/chloroform precipitation of cell lysates and a "liquid phase permethylation" (LPP) procedure. LPP gave better signal-to-noise, yield and precision for most of the glycan nodes from membrane glycoproteins/glycolipids than the conventional solid phase permethylation approach. This GNA approach in cell lysates revealed that specific glycan features such as antennary fucosylation, N-glycan branching, and α2,6-sialylation were elevated in hepatocellular carcinoma (HepG2) cells relative to leukemia cells (THP-1 and K562) and normal donor PBMCs. Additional nodes commonly associated with glycolipids were elevated in the leukemia cells relative to HepG2 cells and PBMCs. Exposure of HepG2 cells to a fucosyltransferase inhibitor resulted in a significant reduction in the relative abundance of 3,4-substituted GlcNAc, which represents antennary fucosylation-providing further proof-of-concept that downregulation of glycosyltransferase activity is detected by shifts in glycan node expression-now detectable in membrane glycoproteins.
Assuntos
Clorofórmio , Leucemia , Humanos , Regulação para Baixo , Glicolipídeos , Glicoproteínas de MembranaRESUMO
Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.
Assuntos
Lasers , Proteínas , Humanos , Cristalografia por Raios X , Proteínas/química , Injeções , NAD(P)H Desidrogenase (Quinona)RESUMO
With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.
RESUMO
Understanding cell-to-cell variation at the molecular level provides relevant information about biological phenomena and is critical for clinical and biological research. Proteins carry important information not available from single-cell genomics and transcriptomics studies; however, due to the minute amount of proteins in single cells and the complexity of the proteome, quantitative protein analysis at the single-cell level remains challenging. Here, we report an integrated microfluidic platform in tandem with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the detection and quantification of targeted proteins from small cell ensembles (> 10 cells). All necessary steps for the assay are integrated on-chip including cell lysis, protein immunocapture, tryptic digestion, and co-crystallization with the matrix solution for MALDI-MS analysis. We demonstrate that our approach is suitable for protein quantification by assessing the apoptotic protein Bcl-2 released from MCF-7 breast cancer cells, ranging from 26 to 223 cells lysed on-chip (8.75 nL wells). A limit of detection (LOD) of 11.22 nM was determined, equivalent to 5.91 × 107 protein molecules per well. Additionally, the microfluidic platform design was further improved, establishing the successful quantification of Bcl-2 protein from MCF-7 cell ensembles ranging from 8 to 19 cells in 4 nL wells. The LOD in the smaller well designs for Bcl-2 resulted in 14.85 nM, equivalent to 3.57 × 107 protein molecules per well. This work shows the capability of our approach to quantitatively assess proteins from cell lysate on the MIMAS platform for the first time. These results demonstrate our approach constitutes a promising tool for quantitative targeted protein analysis from small cell ensembles down to single cells, with the capability for multiplexing through parallelization and automation.
Assuntos
Microfluídica , Proteoma , Limite de Detecção , Proteínas Proto-Oncogênicas c-bcl-2 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.
RESUMO
Increasing evidence has demonstrated that cells are individually heterogeneous. Advancing the technologies for single-cell analysis will improve our ability to characterize cells, study cell biology, design and screen drugs, and aid cancer diagnosis and treatment. Most current single-cell protein analysis approaches are based on fluorescent antibody-binding technology. However, this technology is limited by high background and cross-talk of multiple tags introduced by fluorescent labels. Stable isotope labels used in mass cytometry can overcome the spectral overlap of fluorophores. Nevertheless, the specificity of each antibody and heavy-metal-tagged antibody combination must be carefully validated to ensure detection of the intended target. Thus, novel single-cell protein analysis methods without using labels are urgently needed. Moreover, the labeling approach targets already known motifs, hampering the discovery of new biomarkers relevant to single-cell population variation. Here, we report a combined microfluidic and matrix-assisted laser desorption and ionization (MALDI) mass spectrometric approach for the analysis of protein biomarkers suitable for small cell ensembles. All necessary steps for cell analysis including cell lysis, protein capture, and digestion as well as MALDI matrix deposition are integrated on a microfluidic chip prior to the downstream MALDI-time-of-flight (TOF) detection. For proof of principle, this combined method is used to assess the amount of Bcl-2, an apoptosis regulator, in metastatic breast cancer cells (MCF-7) by using an isotope-labeled peptide as an internal standard. The proposed approach will eventually provide a new means for proteome studies in small cell ensembles with the potential for single-cell analysis and improve our ability in disease diagnosis, drug discovery, and personalized therapy.
Assuntos
Microfluídica , Peptídeos , Biomarcadores , Proteoma , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.
Assuntos
Cristalografia/instrumentação , Elétrons , Dispositivos Lab-On-A-Chip , Lasers , Aldeído Liases/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , HidrodinâmicaRESUMO
The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.
Assuntos
Proteínas de Bactérias/química , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Fotorreceptores Microbianos/química , Conformação Proteica , Luz , Modelos Moleculares , Fatores de TempoRESUMO
The world's first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.
Assuntos
Elétrons , Lasers , Proteínas de Membrana/química , Cristalografia , Cianobactérias/metabolismo , Modelos Moleculares , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/isolamento & purificação , Eletricidade Estática , Síncrotrons , Thermosynechococcus , Raios XRESUMO
The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X-ray free-electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X-ray beam using a liquid jet injector. Owing to the pulsed nature of the X-ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two-photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long-term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal-laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates.
RESUMO
Serial femtosecond crystallography (SFX) is a powerful technique that uses X-ray free-electron lasers (XFEL) to determine structures of biomolecular complexes. Specifically, it benefits the study of atomic resolution structures of large membrane protein complexes and time-resolved reactions with crystallography. One major drawback of SFX studies with XFELs is the consumption of large amounts of a protein crystal sample to collect a complete X-ray diffraction data set for high-resolution crystal structures. This increases the time and resources required for sample preparation and experimentation. The intrinsic pulsed nature of all current X-ray sources is a major reason why such large amounts of sample are required. Any crystal sample that is delivered in the path of the X-ray beam during its "off-time" is wasted. To address this large sample consumption issue, we developed a 3D printed microfluidic system with integrated metal electrodes for water-in-oil droplet generation to dynamically create and manipulate aqueous droplets. We demonstrate on-demand droplet generation using DC potentials and the ability to tune the frequency of droplet generation through the application of AC potentials. More importantly, to assist with the synchronization of droplets and XFEL pulses, we show that the device can induce a phase shift in the base droplet generation frequency. This novel approach to droplet generation has the potential to reduce sample waste by more than 95% for SFX experiments with XFELs performed with liquid jets and can operate under low- and high-pressure liquid injection systems.