Sucrose Density Gradient Centrifugation Research Articles

Potent Protective Immune Responses to Senecavirus Induced by Virus-Like Particle Vaccine in Pigs.

Senecavirus A (SVA) is the pathogen that has recently caused porcine idiopathic vesicular disease (PIVD). The clinical symptoms of PIVD are similar to those of acute foot-and-mouth disease and also can result in the death of newborn piglets, thus entailing economic losses. Vaccine immunization is the most effective way to prevent and control SVA. Among all SVA vaccines reported, only the SVA inactivated vaccine has been successfully developed. However, to ensure the elimination of this pathogen, safer and more effective vaccines are urgently required. A virus-like particles (VLPs)-based vaccine is probably the best alternative to inactivated vaccine. To develop an SVA VLPs vaccine and evaluate its immune effect, a prokaryotic expression system was used to produce SVA capsid protein and assemble VLPs. The VLPs were characterized by affinity chromatography, sucrose density gradient centrifugation, ZetaSizer and transmission electron microscopy. Meanwhile, the SVA CH-HB-2017 strain was used to infect pigs and to determine infection routes and dose. Experimental pigs were then immunized with the SVA VLPs vaccine emulsified in an ISA 201 adjuvant. The results showed that the VLPs vaccine induced neutralizing and specific antibodies at similar levels as an inactivated SVA vaccine after immunization. The level of INF-γ induced by the VLPs vaccine gradually decreased—similar to that of inactivated vaccine. These results indicated that VLPs vaccine may simultaneously cause both cellular and humoral immune responses. Importantly, after the challenge, the VLPs vaccine provided similar levels of protection as the inactivated SVA vaccine. In this study, we successfully obtained novel SVA VLPs and confirmed their highly immunogenicity, thus providing a superior candidate vaccine for defense and elimination of SVA, compared to the inactivated vaccine.

Derlin-3 Is Required for Changes in ERAD Complex Formation under ER Stress.

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a quality control system that induces the degradation of ER terminally misfolded proteins. The ERAD system consists of complexes of multiple ER membrane-associated and luminal proteins that function cooperatively. We aimed to reveal the role of Derlin-3 in the ERAD system using the liver, pancreas, and kidney obtained from different mouse genotypes. We performed coimmunoprecipitation and sucrose density gradient centrifugation to unravel the dynamic nature of ERAD complexes. We observed that Derlin-3 is exclusively expressed in the pancreas, and its deficiency leads to the destabilization of Herp and accumulation of ERAD substrates. Under normal conditions, Complex-1a predominantly contains Herp, Derlin-2, HRD1, and SEL1L, and under ER stress, Complex-1b contains Herp, Derlin-3 (instead of Derlin-2), HRD1, and SEL1L. Complex-2 is upregulated under ER stress and contains Derlin-1, Derlin-2, p97, and VIMP. Derlin-3 deficiency suppresses the transition of Derlin-2 from Complex-1a to Complex-2 under ER stress. In the pancreas, Derlin-3 deficiency blocks Derlin-2 transition. In conclusion, the composition of ERAD complexes is tissue-specific and changes in response to ER stress in a Derlin-3-dependent manner. Derlin-3 may play a key role in changing ERAD complex compositions to overcome ER stress.

Preparation of divalent antigen-displaying enveloped virus-like particles using a single recombinant Bombyx mori nucleopolyhedrovirus bacmid in silkworms

Silkworms have been used as a host for the production of recombinant proteins in a baculovirus expression system using Bombyx mori nucleopolyhedrovirus (BmNPV). To coexpress several recombinant proteins, a silkworm must be coinfected with several recombinant BmNPVs, which requires a difficult DNA manipulation procedure. In this study, we constructed recombinant BmNPVs containing three expression cassettes, Rous sarcoma virus (RSV) Gag protein, surface antigen 1 of Neospora caninum (NcSAG1) and SAG1-related sequence 2 of N. caninum (NcSRS2), by Gibson assembly and the Bac-to-Bac system, designated BmNPV/SAG-SRS-Gag and BmNPV/SAG-Gag-SRS. BmNPV/SAG-SRS-Gag was expressed in silkworms and characterized. NcSAG1 and NcSRS2 were purified with RSV Gag proteins using sucrose density gradient centrifugation and affinity chromatography. RSV Gag formed virus-like particles (RSV-LPs) at a diameter of 20–30 nm based on transmission electron microscopy (TEM). Immuno-TEM analysis showed that both NcSAG1 and NcSRS2 were displayed on the surface of the RSV-LPs. These results indicate that RSV-LPs displaying two different kinds of proteins were produced in the hemolymph of silkworm larvae by the single polycistronic strategy. This expression platform is efficient for generating multiantigen-displaying VLPs and facilitates the development of vaccines against infectious diseases.

Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes.

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

10-Gingerol Targets Lipid Rafts Associated PI3K/Akt Signaling in Radio-Resistant Triple Negative Breast Cancer Cells.

10-Gingerol is a major phenolic lipid found in the rhizomes of ginger (Zingiber officinale). Being amphiphilic in nature, phenolic lipids have the ability to incorporate into cell membranes and modulate membrane properties. The purpose of the present study was to evaluate the effects of 10-gingerol on lipid raft/membrane raft modulation in radio-resistant triple negative breast cancer (MDA-MB-231/IR) cells. The effects of 10-gingerol on MDA-MB-231/IR cells’ proliferation, clonogenic growth, migration, and invasion were assayed using MTT, colony formation, cell migration, and invasion assays, respectively. Sucrose density gradient centrifugation was used to extract lipid rafts. Western blotting and immunofluorescence were employed to assess the effects of 10-gingerol on lipid raft/membrane raft modulation and lipid rafts-associated PI3K/Akt signaling. Cholesterol measurements were carried out using a commercially available kit. 10-gingerol suppressed the proliferation, migration, invasion, and induced apoptosis through targeting the PI3K/Akt signaling pathway in MDA-MB-231/IR cells. Moreover, 10-gingerol was found to modulate the lipid rafts of MDA-MB-231/IR cells and attenuate the key PI3K/Akt signaling components in lipid rafts. The cholesterol content of the lipid rafts and rafts-resident Akt signaling were also affected by exposure to 10-gingerol. The results of the present study highlight rafts-associated PI3K/Akt signaling as a new target of 10-gingerol in MDA-MB-231/IR cells, thus rationalizing a new rafts-mediated treatment approach for radio-resistant triple negative breast cancer cells.

The proteorhodopsins of the dinoflagellate Oxyrrhis marina: ultrastructure and localization by immunofluorescence light microscopy and immunoelectron microscopy

At least 7 proteorhodopsin sequences of Oxyrrhis marina were recently proven in bands obtained by sucrose density gradient centrifugation, and MS analyses revealed that the bands consisted almost of pure, native proteorhodopsins (Rhiel et al. 2020). The proteorhodopsin fractions, i.e., bands B2, B3, and B4 were subjected to transmission electron microscopy. Negative staining revealed that band B2 consisted most likely of monomeric/oligomeric proteorhodopsins with particle dimensions of about 6 nm. Negative staining, freeze-fracture, and cryo-transmission electron microscopy revealed that bands B3 and B4 consisted of vesicular, sheet-like, and cup-shaped structures which all seemed to be composed of protein. Frequently, ring-like protein aggregates were registered at higher magnifications. They measured about 4 nm in diameter with a tiny hole of 1.5 nm in the middle. The bands B2, B3, and B4 were pooled and used to raise an antiserum. Immunoelectron microscopy resulted in intense labeling of the isolated structures. Immunofluorescence light microscopy of formaldehyde-fixed Oxyrrhis cells resulted in intense labeling of the cell periphery. Some cell internal structures became labeled, too. Immunoelectron microscopy of freeze-fractured cells revealed that most likely the membranes of the amphiesmal vesicles were labeled at the cell periphery, while the cell internal label seemed to originate from the food vacuoles.

The protective effects of human umbilical cord mesenchymal stem cell-derived extracellular vesicles on cisplatin-damaged granulosa cells

ObjectiveLong term exposure to gonadotoxic chemotherapy is becoming a major cause of premature ovarian failure/insufficiency (POF/POI) with the increasing cancer incidence among young women. The present study was designed to investigate the protective effects of human cord mesenchymal stem cells (HUCMSCs)-derived extracellular vesicles (EVs) on cisplatin (CDDP)-damaged granulosa cells (GCs) in vitro. Materials and methodsEVs were obtained from supernatant of cultured HUCMSCs by ultracentrifugation method, purified by Sucrose density gradient centrifugation, and then were co-cultured with cisplatin-damaged GCs of 3-weeks female Sprague–Dawley (SD) rats. PKH26 labeled EVs could be observed in normal and CDDP-damaged GCs after 6 h co-culture. ResultsThe surviving GCs were significantly higher and apoptotic GCs were significantly lower in EVs + CDDP group compared with CDDP group. Meanwhile, the levels of E2 and StAR (the key gene related to synthesis of steroid hormone) were significantly higher in EVs + CDDP group compared with CDDP group. Furthermore, the mRNA expression of Caspase 3 was down-regulated significantly and the ratio of Bcl-2/Bax was up-regulated significantly in EVs + CDDP group. Moreover, the protective effect of EVs on CDDP-damaged GCs showed a dose-dependent effect. ConclusionHUCMSCs-derived EVs could become incorporated to CDDP-damaged GCs, and increase the number of living cells, therefore playing important roles in promoting resistance to cisplatin-induced GCs apoptosis and restoring synthesis and secretion of steroid hormone in GCs. This study might provide a theoretical and experimental basis for use of mesenchymal stem cells (MSCs) derived EVs instead of MSCs as a cell-free therapeutic strategy for the patients with POI induced by chemotherapeutic agents.

Data on nucleoid-associated proteins isolated from Mycoplasma Gallisepticum in different growth phases.

Mycoplasma gallisepticum (MG) is one of the smallest free-living and self-replicating organisms, it is characterized by lack of cell wall and reduced genome size. As a result of genome reduction, MG has a limited variety of DNA-binding proteins and transcription factors. To investigate the dynamic changes of the proteomic profile of MG nucleoid, that may assist in revealing its mechanisms of functioning, regulation of chromosome organization and stress adaptation, a quantitative proteomic study was performed on MG nucleoids obtained from the cell culture in logarithmic and stationary phases of synchronous growth. MG cells were grown on a liquid medium with a 9 h starvation period. Nucleoids were obtained from the cell culture at the 26th and the 50th hour (logarithmic and stationary growth phases respectively) by sucrose density gradient centrifugation. LC-MS analysis was carried out on an Ultimate 3000 RSLCnano HPLC system connected to a Fusion Lumos mass spectrometer, controlled by XCalibur software (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). For comprehensive peptide library generation one sample from each biological replicate was run in DDA mode. Then, all the samples were run in a single LC-MS DIA run. Identification of DDA files and DIA quantitation was performed with MaxQuant and Skyline software, correspondingly. All raw data generated from IDA and DDA acquisitions are presented in the PRIDE database with identifier PXD019077.

Proximal Biotinylation-Based Combinatory Approach for Isolating Integral Plasma Membrane Proteins.

Comprehensive profiling of the cell-surface proteome has been challenging due to the lack of tools for an effective and reproducible way to isolate plasma membrane proteins from mammalian cells. Here we employ a proximity-dependent biotinylation approach to label and isolate plasma membrane proteins without an extra in vitro labeling step, which we call Plasma Membrane-BioID. The lipid-modified BirA* enzyme (MyrPalm BirA*) was targeted to the inner leaflet of the plasma membrane, where it effectively biotinylated plasma membrane proteins. Biotinylated proteins were then affinity-purified and analyzed by mass spectrometry. Our analysis demonstrates that combining conventional sucrose density gradient centrifugation and Plasma Membrane-BioID is ideal to overcome the inherent limitations of the identification of integral membrane proteins, and it yields highly pure plasma components for downstream proteomic analysis.

Targeted Myoglobin Delivery as a Strategy for Enhancing the Sensitivity of Hypoxic Cancer Cells to Radiation.

SummaryThe effectiveness of cancer radiotherapy is frequently hindered by the hypoxia of the tumor microenvironment. Direct delivery of oxygen to hypoxic tumor tissues is an attractive strategy to overcome this hypoxia-associated radioresistance. Herein, we report the generation of submicron-sized particles comprising myoglobin fused to the crystal-forming domain of Cry3Aa protein for the targeted delivery of oxygen to cancer cells. We demonstrate that myoglobin-containing particles were successfully produced in Bacillus thuringiensis with the assistance of the Cry3Aa domain I. Furthermore, these particles could be genetically modified to incorporate the cell penetrating peptide TAT and cell targeting peptide A549.1, resulting in particles that exhibited improved cellular uptake and targeting toward A549 cells. Notably, these myoglobin-containing particles increased the intracellular oxygen levels of A549 cells and thereby sensitized them to radiation. These findings suggest that the targeted delivery of O2-bound myoglobin could be an effective approach to enhance the efficacy of radiotherapy.

Development of Sucrose Density Gradient Centrifugation Purification Process for Rabies Vaccine

Development of Sucrose Density Gradient Centrifugation Purification Process for Rabies Vaccine

Delaying osteoarthritis progression through chondrocyte-targeted delivery of WIKI4 using chondrocyte-derived exosomes

Background & Aim Osteoarthritis (OA) is a troublesome disorder that results in the progressive breakdown of the cartilage extracellular matrix. Due to the poor regenerative ability of chondrocytes, OA treatment is difficult. Meanwhile, a recent study showed that tankyrase is a target locus for treating OA, and a selective tankyrase inhibitor, WIKI4, has shown the ability to delay the progression of cartilage degeneration. However, tankyrase inhibitors have serious side effects outside the cartilage, like bone loss. To reduce the side effects, we targeted WIKI4 delivery into cartilage, by loading WIKI4 into exosomes derived from chondrocytes (cExos). Methods, Results & Conclusion The cExos were isolated using sucrose density gradient centrifugation, and they were identified through transmission electron microscopy (TEM), dynamic light scattering (DLS), and western blotting (WB). WIKI4 was loaded, through electroporation, into cExos (WIKI4-cExos), which were tracked through DiI staining. The transfection efficiency into synovial fibroblasts (SFs), chondrocytes, and bone marrow stem cells (BMSCs) were observed, and the OA-related gene expression was measured using WB, qPCR, and ELISA. The effect of WIKI4-cExos on calcification was evaluated based on osteogenic induction and a destabilization of the medial meniscus (DMM) model was used to evaluate the protective effect of WIKI4-cExos. WIKI4-cExos injection commenced the day after surgery and every 7 days subsequently; normal controls underwent sham operation, while DMM models who received normal saline at the same volume as that of the WIKI4-Exos injected models served as the negative controls. cExos without WIKI4 (Con-cExos) also served as controls. The knee joints were obtained 8 weeks post-DMM surgery. Safranine O staining was used for evaluating specimen slices. DiI tracing showed that cExos preferred to transfer into chondrocyte than SFs or BMSCs. WIKI4-cExos significantly increased SOX9, COL-II, and ACAN expression when RUNX2, β-catenin, COL-I, MMP13, and ADAMTS5 expression decreased. During osteogenic induction, WIKI4-cExos significantly decreased chondrocyte calcification without any notable effects on BMSCs. Safranine O staining showed that WIKI4-cExos significantly delayed OA progression, while Con-cExos were not significantly different from normal saline. Cartilage targeting and tankyrase-inhibiting WIKI4-cExos protect cartilage and delay OA progression. This targeting ability also reduced the side effects observed previously outside chondrocytes.

Using biopolymer bodies for encapsulation of hydrophobic products in bacterium

Producing some small hydrophobic molecules in microbes is challenging. Often these molecules cannot cross membranes, and thus their production may be limited by lack of storage space in the producing organism. This study reports a new technology for in vivo storage of valuable hydrophobic products in/on biopolymer bodies in Escherichia coli. A biodegradable and biocompatible polyester – poly (3-hydroxybutyrate) (PHB) – was selected as the intracellular storage vessel to encapsulate lycopene, which is a chromogenic model compound. The hydrophobic interaction between lycopene and PHB was verified by using in vitro binding test and sucrose density gradient centrifugation. Further in vivo characterization was performed by using Confocal Laser Scanning Microscopy (CLSM). The images validated the in vivo co-localization between PHB granules and lycopene. The images also showed that lycopene aggregated in bacteria that did not produce PHB, which may challenge the commonly accepted hypothesis that most lycopene molecules are stored in cell membranes of recombinant host. We also confirmed that producing PHB did not negatively affect lycopene biosynthesis in the E. coli strains and collected data suggesting that PHB titer and lycopene titer were positively correlated when the cells were engineered to co-produce them. The biopolymers that encapsulated hydrophobic molecules could have many useful applications, especially in controlled release because the polymers are biodegradable, and the encapsulated products would be released during the polymer degradation.

Development of pepper vein banding virus chimeric virus-like particles for potential diagnostic and therapeutic applications.

Monoclonal antibodies have attracted wide attention in therapeutics owing to their high efficacy, low toxicity, and specific targeting. However, antibodies cannot cross the cell membrane barrier. Therefore, their therapeutic potential is limited to surface-exposed antigens or secreted proteins. In the present investigation, we have developed a chimeric virus-like particle (VLP) of pepper vein banding virus (PVBV) and explored the possibility of using it as a delivery vehicle for antibodies against intracellular antigens as well as for future applications in immunodiagnostics. The chimeric PVBV particles were generated by genetically engineering the B domain of Staphylococcus aureus protein A (SpA) at the N-terminus of the PVBV coat protein (CP). The chimeric VLPs purified by sucrose density gradient centrifugation had ~440-fold higher affinity towards IgG antibody when compared to SpA. Interestingly, the unassembled chimeric CP with the B-domain at the N-terminus (BCP) purified by Ni-NTA chromatography was a monomer, and it had ~45-fold higher affinity towards antibodies compared to SpA. Additionally, the chimeric particles were able to bind and deliver antibodies against both intracellular (α-tubulin) and surface-exposed antigens (CD 20). However, the BCP monomer failed to enter mammalian cells. Thus, for the first time, we have demonstrated that the assembled VLPs are essential for internalization. These results demonstrate the potential of the use of chimeric PVBV VLPs in diagnostics and, more importantly, as nanocarriers for intracellular delivery of antibodies.

A2E Distribution in RPE Granules in Human Eyes.

A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.

Intensity Ratio of LH2 Complexes from Rhodopseudomonas palustris and Rhodobacter sphaeroides for Purity Determination

Purification procedures to obtain light-harvesting complexes were performed on two photosynthetic bacteria, i.e. Rhodospseudomonas (Rps.) palustris and Rhodobacter (Rb.) sphaeroides. In this study, purification was initially carried out using series of centrifugation towards the bacteria cell to acquire chromatophore of the bacteria. Application of detergent (lauryldimethylamine oxide, or LDAO) to the chromatophore enabled the extraction of membrane containing photosynthetic apparatus. Further purification involved the application of sucrose density gradient centrifugation and ion exchange chromatography. The purity of collected LH2 complexes can be determined by calculating the ratio of AB850 : Aprotein. The resulting LH2 complexes from both bacteria showed relatively high ratio suggesting the purity of the complexes.

A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina.

For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X-100. Subsequent sucrose density gradient centrifugation resulted in three or four red-colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514-516 nm with shoulders toward shorter wavelengths (470-490 nm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in-gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified.

Grad-cryo-EM: Tool to Isolate Translation Initiation Complexes from Rabbit Reticulocyte Lysate Suitable for Structural Studies.

Since its development, single-particle cryogenic electron microscopy (cryo-EM) has played a central role in the study at medium resolution of both bacterial and eukaryotic ribosomal complexes. With the advent of the direct electron detectors and new processing software which allow obtaining structures at atomic resolution, formerly obtained only by X-ray crystallography, cryo-EM has become the method of choice for the structural analysis of the translation machinery. In most of the cases, the ribosomal complexes at different stages of the translation process are assembled in vitro from purified components, which limit the analysis to previously well-characterized complexes with known factors composition. The initiation phase of the protein synthesis is a very dynamic process during which several proteins interact with the translation apparatus leading to the formation of a chronological series of initiation complexes (ICs). Here we describe a method to isolate ICs assembled on natural in vitro transcribed mRNA directly from rabbit reticulocyte lysate (RRL) by sucrose density gradient centrifugation . The Grad-cryo-EM approach allows investigating structures and composition of intermediate ribosomal complexes prepared in near-native condition by cryo-EM and mass spectrometry analyses. This is a powerful approach, which could be used to study translation initiation of any mRNAs, including IRES containing ones, and which could be adapted to different cell extracts.

Polysome Profiling and Metabolic Labeling Methods to Measure Translation in Trypanosoma brucei.

The amount of a protein that is made in a cell is determined not only by the corresponding mRNA level but also by the efficiency with which the mRNA is translated. Very powerful transcriptome-wide methods are available to analyze both the density of ribosomes on each mRNA and the rate at which polypeptides are elongated. However, for many research questions, simpler, less expensive methods are more suitable. Here we describe two methods to assess the general translation status of cells: polysome profiling by sucrose density gradient centrifugation and metabolic labeling using radioactive amino acids. Both methods can also be used to examine translation of individual mRNAs.

A circulating extracellular vesicles-based novel screening tool for colorectal cancer revealed by shotgun and data-independent acquisition mass spectrometry

ABSTRACT Background: Early screening for colorectal cancer (CRC) is essential to improve its prognosis. Liquid biopsies are increasingly being considered for diagnosing cancer due to low invasiveness and high reproducibility. In addition, circulating extracellular vesicles (crEVs, extracellular vesicles isolated from plasma) expressing tumour-specific proteins are potential biomarkers for various cancers. Here, we present a data-independent acquisition (DIA)-mass spectrometry (MS)-based diagnostic method for liquid biopsies. Methods: Extracellular vesicles (EVs) were isolated from culture supernatants of human CRC cell lines, and plasma of patients with CRC at different tumour stages, by overnight ultracentrifugation coupled with sucrose density gradient centrifugation. Tumour-specific EV proteins were prioritized using Tandem Mass Tag (TMT)-based shotgun proteomics and phosphoproteomics. The results were verified in a second independent cohort and a mouse tumour-bearing model using Western blotting (WB). The candidate biomarkers were further validated in a third cohort by DIA-MS. Finally, the DIA-MS methodology was accelerated to permit high-throughput detection of EV biomarkers in another independent cohort of patients with CRC and healthy controls. Results: High levels of total and phosphorylated fibronectin 1 (FN1) in crEVs, haptoglobin (HP), S100A9 and fibrinogen α chain (FGA) were significantly associated with cancer progression. FGA was the most dominant biomarker candidate. Analysis of the human CRC cell lines and the mouse model indicated that FGA+ crEVs were likely released by CRC cells. Furthermore, fast DIA-MS and parallel reaction monitoring (PRM)-MS both confirmed that FGA+ crEVs could distinguish colon adenoma with an area of curve (AUC) in the receiver operating characteristic (ROC) curve of 0.949 and patients with CRC (AUC of ROC is 1.000) from healthy individuals. The performance outperformed conventional tumour biomarkers. The DIA-MS quantification of FGA+ crEVs among three groups agreed with that from PRM-MS. Conclusion: DIA-MS detection of FGA+ crEVs is a potential rapid and non-invasive screening tool to identify early stage CRC. Abbreviations: FGA: fibrinogen α chain; CRC: colorectal cancer; crEVs: circulating extracellular vesicles; EV: extracellular vesicles;MS: mass spectrometry; WB: Western blotting; ROC: receiver operating characteristic; PRM: Parallel Reaction Monitoring; GPC1: Glypican-1; GO: Gene ontology; TEM: transmission electron microscopy; FN1: Fibronectin 1; HP: haptoglobin; TMT: Tandem Mass Tag; LC-MS/MS: liquid chromatography coupled to tandem mass spectrometry; DIA: data-independent acquisition; DDA: data-dependent acquisition; CiRT: Common internal Retention Time standards;AGC: Automatic gain control; AUC: area under curve.