Disposable Columns Research Articles

A Streamlined High-Throughput Plasma Proteomics Platform for Clinical Proteomics with Improved Proteome Coverage, Reproducibility, and Robustness.

Mass spectrometry-based clinical proteomics requires high throughput, reproducibility, robustness, and comprehensive coverage to serve the needs of clinical diagnosis, prognosis, and personalized medicine. Oftentimes these requirements are contradictory to each other. We report the development of a streamlined High-Throughput Plasma Proteomics (sHTPP) platform for untargeted profiling of the blood plasma proteome, which includes 96-well plates and simplified procedures for sample preparation, disposable trap column for peptide loading, robust liquid chromatographic system for separation, data-independent acquisition in tandem mass spectrometry, and DIA-NN, FragPipe, and in-house peptide spectral library-based data analysis. Using the optimized platform at a throughput of 60 samples per day, over 600 protein groups including 57 FDA-approved biomarkers can be consistently identified from whole human plasma, and more than 85% of the detected proteins have 100% completeness in quantitative values across 300 samples. The balance achieved between proteome coverage, throughput, and reproducibility of this sHTPP platform makes it promising in clinical settings, where a large number of samples are to be measured quickly and reliably to support various needs of clinical medicine.

Determination of Ethylene Glycol Dinitrate in Environmental and Forensic Water Samples Using Microextraction by Packed Sorbent Followed by Gas Chromatography

Ethylene glycol dinitrate (EGDN) is a liquid nitrate ester, a secondary explosive. In the past, it was used as an explosive ingredient in dynamite along with nitroglycerine. Due to its various applications, the reliable detection of EGDN in the environment is a key issue for both forensic and environmental applications. In these areas, sensitive and reliable methods for determining the concentration of nitro compounds are needed. Microextraction by packed sorbent (MEPS) is an innovative approach to green technology in the sample preparation field. Compared to conventional solid-phase extraction (SPE), MEPS uses a smaller sample volume and can be easily combined with various chromatographic techniques. An important benefit is the reduction of sorbent amount and up to 100-times repeatable use compared to disposable SPE columns, thus reducing the costs of analysis as well as waste production. Optimal extraction parameters for isolating EGDN from water, e.g., 30 µL of toluene as extraction agent, working in one cycle and in draw/discard mode, were selected. Method validation was performed, obtaining a limit of detection and a limit of quantification of 0.45 pg/μL and 1.34 pg/μL, respectively. Accuracy in terms of recovery rates was evaluated over a wide concentration range, obtaining values from 83.7 to 90.0%. The satisfactory linearity expressed by the coefficient of determination was 0.9914. A matrix factor of −9.3% indicates a weak matrix effect. The application to real environmental water samples and a forensic post-blast wash water sample was realized. EGDN detection in the post-blast samples provides valuable information for forensic technicians.

Evaluation of Disposable Trap Column nanoLC-FAIMS-MS/MS for the Proteomic Analysis of FFPE Tissue.

Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving a high proteomic depth and limiting the overall analysis time. To this end, we evaluated the merits of online coupling of single-use disposable trap column nanoflow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS), and tandem mass spectrometry (nLC-FAIMS-MS/MS). The data show that ≤600 ng of peptide digest should be loaded onto the chromatographic part of the system. Careful characterization of the FAIMS settings enabled the choice of optimal combinations of compensation voltages (CVs) as a function of the employed LC gradient time. We found nLC-FAIMS-MS/MS to be on par with StageTip-based off-line basic pH reversed-phase fractionation in terms of proteomic depth and reproducibility of protein quantification (coefficient of variation ≤15% for 90% of all proteins) but requiring 50% less sample and substantially reducing sample handling. Using FFPE materials from the lymph node, lung, and prostate tissue as examples, we show that nLC-FAIMS-MS/MS can identify 5000-6000 proteins from the respective tissue within a total of 3 h of analysis time.

A single column separation method for barium isotope analysis of geologic and hydrologic materials with complex matrices

The increasing significance of barium (Ba) in environmental and geologic research in recent years has led to interest in the application of the Ba isotopic composition as a tracer for natural materials with complex matrices. Most Ba isotope measurement techniques require separation of Ba from the rest of sample prior to analysis. This paper presents a method using readily available materials and disposable columns that effectively separates Ba from a range of geologic and hydrologic materials, including carbonate minerals, silicate rocks, barite, river water, and fluids with high total dissolved solids and organic content such as oil and gas brines, rapidly and without need for an additional cleanup column. The technique involves off-the-shelf columns and cation exchange resin and a two-reagent elution that uses 2.5 N HCl followed by addition of 2.0 N HNO3. We present data to show that major matrix elements from almost any natural material are separated from Ba in a single column pass, and that the method also effectively reduces or eliminates isobaric interferences from lanthanum and cerium.

Gamma irradiating chromatography columns enables bioburden-free integrated continuous biomanufacturing.

An important consideration for integrated continuous biomanufacturing is that the downstream chromatography steps integrated with the bioreactor should maintain a low bioburden state throughout the entire duration of the operation. One potential strategy to achieve this is to start bioburden-free and functionally close the chromatography system. While chromatography skids themselves can be rendered bioburden-free, limitations exist in applying these methods to chromatography columns. The small column sizes used in continuous multicolumn chromatography enable gamma irradiation of disposable columns to render them bioburden-free. However, this approach has not been widely implemented, likely because gamma irradiation can negatively impact resin performance. Here, several protective mobile-phase modifiers were screened and shown to help chromatography resins retain naïve-like performance. Gamma irradiated columns were then integrated into perfusion bioreactors for continuous capture. Successful integrated continuous capture downstream of perfusion bioreactors for greater than 40 days using protein A, custom affinity, and non-affinity capture resins for multiple biologic modalities is demonstrated in development and commercial settings. No indications of time-based performance decline or bioburden growth have been observed. This strategy enables bioburden-free integrated continuous biomanufacturing operations and could allow full process closure and decreased environmental control requirements for facilities; thus, permitting simultaneous multi-product operations in a ballroom arrangement.

Development of a low-cost disposable bioreactor for pilot scale production of Hypericum perforatum L. adventitious roots

Bioreactors are widely used for industrial production of therapeutic products. In this study, a disposable bubble column bioreactor made from flexible plastic films and cheap accessories was developed and its applicability for plant root culture was tested. Hypericum perforatum adventitious roots were cultivated in a set of 15, 30, and 60-L bioreactors for a four weeks period and 14.4, 11, and 10 g dry roots per liter were harvested respectively. Appropriate air flow pattern caused the roots to distribute to more than 70 % of the liquid culture volume without using any support matrix. The packing densities showed 30 % variation at different location of root biomass. Determination of hypericins, total phenols, and total flavonoids indicated some variation throughout the bioreactor, however, the average of them resembled the roots cultivated in flasks. Appropriate bioreactors remain an obstacle in commercialization of plant root cultures and the developed bioreactor seems appropriate for mass production of some adventitious and hairy roots in a modular design.

The screening of organophosphorus, organochlorine and synthetic pyrethroid pesticides residues in beef fat by tandem solid-phase extraction technique

For regulatory monitoring of pesticides in environmental matrices, a multiresidue analysis through solid phase extraction (SPE) technique and using gas chromatographic determination of organochlorine(OC), organophosphorus(OP) and synthetic pyrethroid pesticides residues in beef fat was studied. The group of pesticides was added to dissolved beef fat (0.5 g) and blended with 8% de-activation florisil. The florisil/fat mixture was packed into disposable filtration columns. This column was tandem with an octadecyl SPE column and was eluted with acetonitrile. The pesticide residues were determined by gas chromatography with electron capture and flame photometric detectors. Three kinds (including OC, OP and synthetic pyrethroid) with a total of 71 pesticides (including their metabolites) were tested in this study. With the exception of isoxthion and flucythrinate with high recovery rates (122% ± 13% and 137% ± 31%, respectively), according to the correlation coefficients for the 71 extracted pesticide standard curves (ranging from 0.9915 ± 0.0176 to 0.9999 ± 0.0200, n = 5), average recovery rate (ranging from 68 ± 9% to 117 ± 11%, n = 25 for each insecticide), inter-assay variability (ranging from 3 ± 3% to 17 ± 12%, n = 25 for each insecticide), intra-assay variability(ranging from 3 to 19%, n = 5 for each insecticide) indicated that the methodology is acceptable for the extraction, determination, and screening of theses residues in beef fat. The detection limits for OCs, OPs and synthetic pyrethroid pesticides are 2-21 ng/g, 6-31 ng/g and 3-118 ng/g, respectively. Comparing with studies conducted by the National Analytical Reference Laboratory in Western Australia in the field of pesticide residue testing in beef fat, the results indicated that the present method could accurately measure commonly used pesticide chemicals.

A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-robust Proteomics

To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump. This simplified design enables robust operation over thousands of sample injections. Furthermore, the steps between injections are performed in parallel, reducing overhead time to a few minutes and allowing analysis of more than 200 samples per day. From fractionated HeLa cell lysates, deep proteomes covering more than 130,000 sequence unique peptides and close to 10,000 proteins were rapidly acquired. Using this data as a library, we demonstrate quantitation of 5200 proteins in only 21 min. Thus, the new system - termed Evosep One - analyzes samples in an extremely robust and high throughput manner, without sacrificing in depth proteomics coverage.

Scalability of pre-packed preparative chromatography columns with different diameters and lengths taking into account extra column effects

Small pre-packed columns are commonly used to estimate the optimum run parameters for pilot and production scale. The question arises if the experiments obtained with these columns are scalable, because there are substantial changes in extra column volume when going from a very small scale to a benchtop column. In this study we demonstrate the scalability of pre-packed disposable and non-disposable columns of volumes in the range of 0.2–20 ml packed with various media using superficial velocities in the range of 30–500 cm/h. We found that the relative contribution of extra column band broadening to total band broadening was not only high for columns with small diameters, but also for columns with a larger volume due to their wider diameter. The extra column band broadening can be more than 50% for columns with volumes larger than 10 ml. An increase in column diameter leads to high additional extra column band broadening in the filter, frits, and adapters of the columns. We found a linear relationship between intra column band broadening and column length, which increased stepwise with increases in column diameter. This effect was also corroborated by CFD simulation. The intra column band broadening was the same for columns packed with different media. An empirical engineering equation and the data gained from the extra column effects allowed us to predict the intra, extra, and total column band broadening just from column length, diameter, and flow rate.

Column-to-column packing variation of disposable pre-packed columns for protein chromatography

In the biopharmaceutical industry, pre-packed columns are the standard for process development, but they must be qualified before use in experimental studies to confirm the required performance of the packed bed. Column qualification is commonly done by pulse response experiments and depends highly on the experimental testing conditions. Additionally, the peak analysis method, the variation in the 3D packing structure of the bed, and the measurement precision of the workstation influence the outcome of qualification runs. While a full body of literature on these factors is available for HPLC columns, no comparable studies exist for preparative columns for protein chromatography. We quantified the influence of these parameters for commercially available pre-packed and self-packed columns of disposable and non-disposable design. Pulse response experiments were performed on 105 preparative chromatography columns with volumes of 0.2–20ml. The analyte acetone was studied at six different superficial velocities (30, 60, 100, 150, 250 and 500cm/h). The column-to-column packing variation between disposable pre-packed columns of different diameter-length combinations varied by 10–15%, which was acceptable for the intended use. The column-to-column variation cannot be explained by the packing density, but is interpreted as a difference in particle arrangement in the column. Since it was possible to determine differences in the column-to-column performance, we concluded that the columns were well-packed. The measurement precision of the chromatography workstation was independent of the column volume and was in a range of±0.01ml for the first peak moment and±0.007 ml2 for the second moment. The measurement precision must be considered for small columns in the range of 2ml or less. The efficiency of disposable pre-packed columns was equal or better than that of self-packed columns.

A new platform for serological analysis based on porous 3-dimensional polyethylene sinter bodies

A new sensitive and selective platform, three-dimensional immunosensor, has been developed for a rapid serological diagnosis; detection of a Borrelia infection was considered as a model assay. The immunosensor is based on a 3-dimensional (3D) porous solid surface (sinter body) with dimensions of 2×2.5mm where a recombinant variable lipoprotein surface-exposed protein (VlsE; Borrelia-antigen) is immobilized by different techniques. The sinter body served as a robust and inexpensive carrier, which facilitated a successful hydrophobic adsorption as well as covalent immobilization of the antigen with sufficient amounts of on the surface. Because of sinter body’s porosity, the detection could be performed in an immune affinity flow system based on a little disposable plastic column. The flow of reagents through the column is advantageous in terms of reducing the non-specific interaction and shortening the test time. Furthermore, three labels were tested for a colorimetric detection: i) a horseradish peroxidase (HRP) labeled secondary antibody, ii) nanoparticles based on Sudan IV, and iii) gold nanoparticles modified with protein A. HRP secondary labeled antibody provides the most sensitive test, 1000 fold dilution of serum sample can be clearly detected in only 20min. Gold nanoparticles modified with protein A were used as a direct label or as a catalyst for reduction of silver ions. Direct detection with gold nanoparticles provides short time of analysis (5min) while detection of metallic silver required longer time (12min) but with improved sensitivity. Nanoparticles based on Sudan IV showed high background and were less favorable. The assay is distinctive because of the rapid analysis time with all used labels, longest 20min. Compared to classical serological methods for Borrelia diagnosis, the developed method offers a simple, rapid and reliable tool of analysis with minimal cost and can be easily transferred to other infectious diseases.

Automated cassette-based production of high specific activity [203/212Pb]peptide-based theranostic radiopharmaceuticals for image-guided radionuclide therapy for cancer

A method for preparation of Pb-212 and Pb-203 labeled chelator-modified peptide-based radiopharmaceuticals for cancer imaging and radionuclide therapy has been developed and adapted for automated clinical production. Pre-concentration and isolation of radioactive Pb2+ from interfering metals in dilute hydrochloric acid was optimized using a commercially-available Pb-specific chromatography resin packed in disposable plastic columns. The pre-concentrated radioactive Pb2+ is eluted in NaOAc buffer directly to the reaction vessel containing chelator-modified peptides. Radiolabeling was found to proceed efficiently at 85°C (45min; pH 5.5). The specific activity of radiolabeled conjugates was optimized by separation of radiolabeled conjugates from unlabeled peptide via HPLC. Preservation of bioactivity was confirmed by in vivo biodistribution of Pb-203 and Pb-212 labeled peptides in melanoma-tumor-bearing mice. The approach has been found to be robustly adaptable to automation and a cassette-based fluid-handling system (Modular Lab Pharm Tracer) has been customized for clinical radiopharmaceutical production. Our findings demonstrate that the Pb-203/Pb-212 combination is a promising elementally-matched radionuclide pair for image-guided radionuclide therapy for melanoma, neuroendocrine tumors, and potentially other cancers.

Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer

ObjectiveIsolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable.MethodsPlasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells.ResultsExosomes eluting in fractions #3–5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03).ConclusionsMini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications.

Detection of polychlorinated biphenyls in transformer oils in Vietnam by multiphoton ionization mass spectrometry using a far-ultraviolet femtosecond laser as an ionization source

Polychlorinated biphenyls (PCBs) in transformer and food oils were measured using gas chromatography combined with multiphoton ionization mass spectroscopy. An ultrashort laser pulse emitting in the far-ultraviolet region was utilized for efficient ionization of the analytes. Numerous signal peaks were clearly observed for a standard sample mixture of PCBs when the third and fourth harmonic emissions (267 and 200nm) of a femtosecond Ti:sapphire laser (800nm) were employed. The signal intensities were found to be greater when measured at 200nm compared with those measured at 267nm, providing lower detection limits especially for highly chlorinated PCBs at shorter wavelengths. After simple pretreatment using disposable columns, PCB congeners were measured and found to be present in the transformer oils used in Vietnam.

Purification of His-Tagged Proteins.

Ni-NTA affinity purification of His-tagged proteins is a bind-wash-elute procedure that can be performed under native or denaturing conditions. Here, protocols for purification of His-tagged proteins under native, as well as under denaturing conditions, are given. The choice whether to purify the target protein under native or denaturing conditions depends on protein location and solubility, the accessibility of the His tag, and the desired downstream application. His-tagged proteins can be purified by a single-step affinity chromatography, namely immobilized metal ion affinity chromatography (IMAC), which is commercially available in different kinds of formats, Ni-NTA matrices being the most widely used. The provided protocols describe protein purification in the batch binding mode and apply gravity-assisted flow in disposable columns; this procedure is simple to conduct and extremely robust. IMAC purification can equally be performed in prepacked columns using FPLC or other liquid chromatography instrumentation, or using magnetic bead-based methods (Block et al., 2009).

A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional neurotensin receptor 1 variants from E. coli

G protein-coupled receptors (GPCRs) are key players of cell signaling, thus representing important drug targets for the treatment of human diseases. Since inherent difficulties in receptor production and handling have precluded the application of many in vitro experiments, major questions about GPCR mechanisms and dynamics remain elusive to date. We recently used directed evolution in Escherichia coli on neurotensin receptor 1 (NTR1) for the generation of GPCR variants with greatly elevated functional expression levels and with excellent stability in detergent micelles. In this work we outline a highly efficient purification method for our evolved receptor variants, which is based on the application of an inexpensive, disposable high-affinity ligand column as the initial purification step. The ligand resin allows isolation of correctly folded GPCR variants directly from whole E. coli cell lysates at the scale of 10mg and it permits preparations of agonist- and antagonist-bound receptor samples. The purification principle presented here was key to the first structures of signaling-active NTR1 variants (Egloff et al., 2014). Since E. coli is uniquely suitable for the production of fully deuterated proteins, our method provides the basis for an array of NMR experiments that were not feasible for GPCRs to date, but which will shed light on novel aspects of receptor function and dynamics.

Determination of 20 polychlorinated biphenyls in fish samples by gas chromatography-triple-quadrupole mass spectrometry isotope dilution method

An improved method based on the isotope dilution technique was developed for the determination of 20 polychlorinated biphenyls (PCBs) in fish samples, including seven indicator polychlorinated biphenyls. The analysis was performed by gas chromatography-triple-quadrupole mass spectrometry (GC-MS/MS) in the multiple reaction monitoring (MRM) mode. Two MRM transitions were monitored for each PCB congener for quantitation and qualification purposes. The edible parts of the fish samples were shattered, frozen, and dried. 13C-PCB surrogate standards were spiked into the fish samples. The PCBs in fish samples and the surrogate standards were extracted by auto-Soxhlet extraction. The extract was cleaned-up by a disposable multi-layer column packed with acidic silica gel and basic alumina. 13C-PCB recovery standards were spiked into the extract before injection. The separation was performed on a TG-5MS capillary column. Quantitation was performed by the internal standard method. Every group of PCBs used a same number chlorinated 13C-isotope PCBs congener as the internal standard. The calibration curves of 20 PCB congeners were linear (correlation coefficient (r)>0.99) within the range of 0.05-10 μg/L. For the recovery test, three levels were spiked in matrices. The average recoveries obtained by this method were 80.3%-117.6% and the relative standard deviations (RSDs, n=6) were 5.09%-18.5%. The limits of determination (LODs) were 0.01-0.02 μg/kg. Contents of the 20 PCBs in the fish samples were in the range 1.2-8.8 μg/kg (wet weight), while those of the seven indicator PCBs were in the range 0.68-6.4 μg/kg (wet weight). The method is suitable for determining polychlorinated biphenyls in fish samples.

Extraction of trace elements in human fluids using cation exchange resin (chelex-100) columns for ICP-MS analysis

A successful metal ion extraction method was developed using cation exchange resin packed disposable micro pipette tip columns for the extraction of trace metal ions in human fluids. Using this method, concentrations of Mn, Ni, Cu, Zn, Cd and Pb ions in human urine were determined by inductively coupled plasma mass spectroscopy (ICP-MS). The main objective of this study was to develop a technique to overcome spectral interferences in ICP analysis of trace elements in biological fluids mainly due to salts present in the samples. The amount of resins packed to the column, amount of eluent needed to extract trace elements, and particle size of the resin were examined to determine conditions for metal extraction. The validity of this technique was evaluated by calculating recoveries of trace element concentrations of above elements in certified standard urine samples and by using the technique for trace metal spiked urine samples. The results revealed that Pb, Cd, Zn, Cu and Mn had good recoveries (about 95%) while Ni &amp; Co had about 80% recovery. It was also found that the recoveries of trace elements depended on the concentrations of relevant elements in the samples.Journal of the University of Ruhuna 2013 1(2): 23-30

QuiPO : qui administre les EPO en prédialyse ?

Identification of post-translational modifications of proteins in biological samples often requires access to preanalytical purification and concentration methods. In the purification step high or low molecular weight substances can be removed by size exclusion filters, and high abundant proteins can be removed, or low abundant proteins can be enriched, by specific capturing tools. In this paper is described the experience and results obtained with a recently emerged and easy-to-use affinity purification kit for enrichment of the low amounts of EPO found in urine and plasma specimens. The kit can be used as a pre-step in the EPO doping control procedure, as an alternative to the commonly used ultrafiltration, for detecting aberrantly glycosylated isoforms. The commercially available affinity purification kit contains small disposable anti-EPO monolith columns (6 μL volume, Ø7 mm, length 0.15 mm) together with all required buffers. A 24-channel vacuum manifold was used for simultaneous processing of samples. The column concentrated EPO from 20 mL urine down to 55 μL eluate with a concentration factor of 240 times, while roughly 99.7% of non-relevant urine proteins were removed. The recoveries of Neorecormon (epoetin beta), and the EPO analogues Aranesp and Mircera applied to buffer were high, 76%, 67% and 57%, respectively. The recovery of endogenous EPO from human urine was 65%. High recoveries were also obtained when purifying human, mouse and equine EPO from serum, and human EPO from cerebrospinal fluid. Evaluation with the accredited EPO doping control method based on isoelectric focusing (IEF) showed that the affinity purification procedure did not change the isoform distribution for rhEPO, Aranesp, Mircera or endogenous EPO. The kit should be particularly useful for applications in which it is essential to avoid carry-over effects, a problem commonly encountered with conventional particle-based affinity columns. The encouraging results with EPO propose that similar affinity monoliths, with the appropriate antibodies, should constitute useful tools for general applications in sample preparation, not only for doping control of EPO and other hormones such as growth hormone and insulin but also for the study of post-translational modifications of other low abundance proteins in biological and clinical research, and for sample preparation prior to in vitro diagnostics.

SNAREpin Assembly by Munc18-1 Requires Previous Vesicle Docking by Synaptotagmin 1

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.