Project description:Extracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. EVs-based liquid biopsies usually require EVs isolation before content analysis, which frequently increases sample volume requirements. We here present a Flow Cytometry (FC) strategy that does not require isolation or concentration of EVs prior to staining. By doing so, it enables population analysis of EVs in samples characterized by challenging small volumes, while reducing overall sample processing time. To illustrate its application, we performed longitudinal non-lethal population analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By quantifying the proportion of vesicular particles in purified and non-purified biological samples, this method also serves as a precious tool to quality control isolates of EVs purified by different protocols. Our FC strategy has an unexplored clinical potential to analyze EVs in biofluids with intrinsically limited volumes and to multiply the number of different analytes in EVs that can be studied from a single collection of biofluid.
Project description:Turbulent flow chromatography is an online solid phase extraction mode that achieves the extraordinary effect of proxying an upper molecular weight cutoff for the retained molecules, based on loading the sample at high linear velocities. Despite the potential of being a universal sample preparation technique prior to inductively coupled plasma mass spectrometry and liquid chromatography mass spectrometry, it employs specific hardware and expensive consumables. In the present work we apply this technique using off-the-shelf fluidic components and the niche "bead injection" methodology. For the first time, this procedure has been executed with a pressure of approximately 20 bar, compared to the low pressure of the classic setup, achieving a sample throughput >285 h-1 for the SPE/TFC procedure, or 20 h-1 if the procedure involves renewing the sorbent, using no more than 4 mg of sorbent for every μ-SPE. Another novelty is that sorbent packing and unpacking has been controlled with a smart method using real-time pressure feedback as quality control for truly unattended operation. Finally, the turbulent flow chromatography principle has been comprehensively characterized, providing similar performance to that demonstrated in earlier literature, and the ancillary sample preparation capabilities, e.g., in-valve acidification, have been demonstrated by the fractionation of gadolinium in surface waters prior to ICP-MS, an element of increasing surface water concern due to its use as a magnetic resonance contrast agent.
Project description:Multi-omics analysis is a powerful and increasingly utilized approach to gain insight into complex biological systems. One major hindrance with multi-omics, however, is the lengthy and wasteful sample preparation process. Preparing samples for mass spectrometry (MS)-based multi-omics involves extraction of metabolites and lipids with organic solvents, precipitation of proteins, and overnight digestion of proteins. These existing workflows are disparate and laborious. Here, we present a simple, efficient, and unified approach to prepare lipids, metabolites, and proteins for MS analysis. Our approach, termed the Bead-enabled Accelerated Monophasic Multi-omics (BAMM) method, combines an n-butanol-based monophasic extraction with unmodified magnetic beads and accelerated protein digestion. We demonstrate that the BAMM method affords comparable depth, quantitative reproducibility, and recovery of biomolecules as state-of-the-art multi-omics methods (e.g., Matyash extraction and overnight protein digestion). However, the BAMM method only requires about 3 h to perform, which saves 11 steps and 19 h on average compared to published multi-omics methods. Furthermore, we validate the BAMM method for multiple sample types and formats (biofluid, culture plate, and pellet) and show that in all cases, it produces high biomolecular coverage and data quality.
Project description:Nucleic acid (NA) extraction and purification has become a common technique in both research and clinical laboratories. Current methods require repetitive wash steps with a pipet that are laborious and time-consuming, making the procedure inefficient for clinical settings. We present here a simple technique that relies on spontaneous biphasic plug flow inside a capillary to achieve sample preparation. By filling the sample with oil, aqueous contaminants were displaced from the captured NA without pipetting wash buffers or use of external force and equipment. mRNA from mammalian cell culture was purified, and polymerase chain reaction (PCR) amplification showed similar threshold cycle values as those obtained from a commercially available kit. Human immunodeficiency virus (HIV) viral-like particles were spiked into serum and a 5-fold increase in viral RNA extraction yield was achieved compared to the conventional wash method. In addition, viral RNA was successfully purified from human whole blood, and a limit of detection of approximately 14 copies of RNA extracted per sample was determined. The results demonstrate the utility of the current technique for nucleic acid purification for clinical purposes, and the overall approach provides a potential method to implement nucleic acid testing in low-resource settings.
Project description:Complete enzymatic digestion of proteins for bottom-up proteomics is substantially improved by use of detergents for denaturation and solubilization. Detergents however, are incompatible with many proteases and highly detrimental to LC-MS/MS. Recently; filter-based methods have seen wide use due to their capacity to remove detergents and harmful reagents prior to digestion and mass spectrometric analysis. We hypothesized that non-specific protein binding to negatively charged silica-based filters would be enhanced by addition of lyotropic salts, similar to DNA purification. We sought to exploit these interactions and investigate if low-cost DNA purification spin-filters, 'Minipreps,' efficiently and reproducibly bind proteins for digestion and LC-MS/MS analysis. We propose a new method, Miniprep Assisted Proteomics (MAP), for sample preparation. We demonstrate binding capacity, performance, recovery and identification rates for proteins and whole-cell lysates using MAP. MAP recovered equivalent or greater protein yields from 0.5-50 μg analyses benchmarked against commercial trapping preparations. Nano UHPLC-MS/MS proteome profiling of lysates of Escherichia coli had 99.3% overlap vs. existing approaches and reproducibility of replicate minipreps was 98.8% at the 1% FDR protein level. Label Free Quantitative proteomics was performed and 91.2% of quantified proteins had a %CV <20% (2044/2241). Miniprep Assisted Proteomics can be performed in minutes, shows low variability, high recovery and proteome depth. This suggests a significant role for adventitious binding in developing new proteomics sample preparation techniques. MAP represents an efficient, ultra-low-cost alternative for sample preparation in a commercially obtainable device that costs ∼$0.50 (USD) per miniprep.
Project description:We introduced a single-cell proteome microfluidic platform, integrating cell capture, lysis, protein reduction, alkylation and online digestion. The online enzymolysis facilitated the fast, efficient and all-in-one proteomic sample preparation, providing wide dynamic range and enhanced coverage of proteins.
Project description:A huge, unprecedented demand for gelatin coupled with its implications on global sustainability has resulted in the need to discover novel proteins with gelling attributes for applications in the food industry. Currently used gelation assays require large sample volumes and thus the screening for novel gelling proteins is a formidable technical challenge. In this paper, we report the 'Floating Sphere Assay' which is a simple, economical, and miniaturized assay to detect minimum gelling concentration with volumes as low as 50 μl. Results from the Floating Sphere Assay are consistent with currently used methods for gelation tests and accurately estimate the Minimum Gelling Concentrations (MGCs) of gelatin, κ-carrageenan and gellan gum. The assay was also able to differentiate the strengths of strong and weak gellan gum gels prepared at pH 3.5 and pH 7.0 respectively. The Floating Sphere Assay can be utilized in high-throughput screens for gelling proteins and can accelerate the discovery of gelatin substitutes.
Project description:This study was performed to optimize the analytical method for multi-residues of 60 compounds in flatfish samples. Three sample preparation methods were tested to identify the optimal recovery conditions for target analytes. As a result, 10 mL of water/acetonitrile (1:4, v/v) was used to extract analytes from fish samples. For purification, C18 and 10 mL of acetonitrile saturated hexane were used to treat the samples. After evaporation and reconstitution, the fish samples were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. The proposed method was validated according to the CODEX guidelines (CAC/GL-71). Our results showed the recoveries of 73.2%-115% and coefficients of variation of 1.6%-22.1%. The limit of quantification was 0.0005-0.005 mg/kg in the fishery products. In analysis of real samples, no samples exceeded the limit of quantification. This analytical method can be used for multi-residue screening and confirmation of the residues of veterinary drugs in fishery products.