The ELISA is the mainstay for sensitive and quantitative detection of protein analytes. Despite its utility, ELISA is time-consuming, resource-intensive, and infrastructure-dependent, limiting its ...availability in resource-limited regions. Here, we describe a self-contained immunoassay platform (the “D4 assay”) that converts the sandwich immunoassay into a point-of-care test (POCT). The D4 assay is fabricated by inkjet printing assay reagents as microarrays on nanoscale polymer brushes on glass chips, so that all reagents are “on-chip,” and these chips show durable storage stability without cold storage. The D4 assay can interrogate multiple analytes from a drop of blood, is compatible with a smartphone detector, and displays analytical figures of merit that are comparable to standard laboratory-based ELISA in whole blood. These attributes of the D4 POCT have the potential to democratize access to high-performance immunoassays in resource-limited settings without sacrificing their performance.
Single-cell proteomics (SCP) has great potential to advance biomedical research and personalized medicine. The sensitivity of such measurements increases with low-flow separations (<100 nL/min) due ...to improved ionization efficiency, but the time required for sample loading, column washing, and regeneration in these systems can lead to low measurement throughput and inefficient utilization of the mass spectrometer. Herein, we developed a two-column liquid chromatography (LC) system that dramatically increases the throughput of label-free SCP using two parallel subsystems to multiplex sample loading, online desalting, analysis, and column regeneration. The integration of MS1-based feature matching increased proteome coverage when short LC gradients were used. The high-throughput LC system was reproducible between the columns, with a 4% difference in median peptide abundance and a median CV of 18% across 100 replicate analyses of a single-cell-sized peptide standard. An average of 621, 774, 952, and 1622 protein groups were identified with total analysis times of 7, 10, 15, and 30 min, corresponding to a measurement throughput of 206, 144, 96, and 48 samples per day, respectively. When applied to single HeLa cells, we identified nearly 1000 protein groups per cell using 30 min cycles and 660 protein groups per cell for 15 min cycles. We explored the possibility of measuring cancer therapeutic targets with a pilot study comparing the K562 and Jurkat leukemia cell lines. This work demonstrates the feasibility of high-throughput label-free single-cell proteomics.
Due to sensitivity limitations, global proteome measurements generally require large amounts of biological starting material, which masks heterogeneity within the samples and differential protein ...expression among constituent cell types. Methods for spatially resolved proteomics are being developed to resolve protein expression for distinct cell types among highly heterogeneous tissues, but have primarily been applied to mammalian systems. Here we evaluate the performance of cell-type-specific proteome analysis of tomato fruit pericarp tissues by a platform integrating laser-capture microdissection (LCM) and a recently developed automated sample preparation system (nanoPOTS, nanodroplet processing in one pot for trace samples). Tomato fruits were cryosectioned prior to LCM and tissues were dissected and captured directly into nanoPOTS chips for processing. Following processing, samples were analyzed by nanoLC-MS/MS. Approximately 1900 unique peptides and 422 proteins were identified on average from ∼0.04 mm2 tissues comprising ∼8–15 parenchyma cells. Spatially resolved proteome analyses were performed using cells of outer epidermis, collenchyma, and parenchyma. Using ≤200 cells, a total of 1,870 protein groups were identified and the various tissues were easily resolved. The results provide spatial and tissue-specific insights into key enzymes and pathways involved in carbohydrate transport and source–sink relationships in tomato fruit. Of note, at the time of fruit ripening studied here, we identified differentially abundant proteins throughout the pericarp related to chlorophyll biogenesis, photosynthesis, and especially transport.
Background
Many healthcare facilities and providers prohibit blenderized tube feeding (BTF) for patients who request it due to concerns of high microbial load. The current project compared microbial ...loads of a standard ready‐to‐feed polymeric commercial formula (CF), a BTF made using baby food (BTF‐BF), and a BTF prepared from blending whole food (BTF‐WF), following food safety standards expected of U.S. hospitals.
Methods
Three tube‐feeding formulas (CF, BTF‐BF, BTF‐WF) were prepared in a U.S. hospital and delivered in vitro to an unoccupied patient room. Samples were collected at zero hour, 2 hours, and 4 hours and compared for growth of aerobic microorganisms, Staphylococus aureus, coliforms, and Escherichia coli. The experiment was conducted in triplicate, 1 week apart.
Results
No S. aureus or coliform/E. coli were detected at any time point following preparation, and total bacterial count was well below acceptable limits. All 3 feeding formulas at zero hour, 2 hours, and 4 hours for each of the 3 sampling dates were acceptable for human consumption.
Conclusion
Judicious BTF recipe selection and adherence to safe food handling provide a safe feeding substrate equivalent to CF in the hospital setting. Due to increased use and interest in BTF by patients and their caregivers, healthcare facilities may need to reexamine their policies prohibiting BTF use.
Objective: The purpose of this study was to examine the effect of a fatigue protocol on Vestibular/Ocular Motor Screening (VOMS) performance.
Design: Within subjects, repeated measures, crossover.
...Methods: Fifteen healthy, physically-active participants (22.20 ± 1.424 years) completed 2 sessions under 2 conditions. A pretest VOMS, condition protocol, and a posttest VOMSwere performed. The control condition consisted of rest, while the experimental consisted of a fatigue protocol.
Results: The primary outcome measures were VOMS performance scores and Near Point of Convergence (NPC) measurements. Statistically significant interaction effects for NPC, F(1,14) = 9.38, p = .008, and total VOMS score, F(1,14) = 10.96, p = .005 were observed. For NPC, posttest (9.12 ± 4.99 cm) was significantly different, t(14) = −2.60, p = .021, than pretest scores (7.12 ± 3.19 cm). For total VOMS score, posttest experimental scores (4.93 ± 5.12) were significantly different, t(14) = −3.06, p = .009, than pretest severity scores (1.73 ± 3.67).
Conclusions: Significant increases were found in total VOMS and NPC scores following exertional fatigue. Exertional fatigue affects symptoms associated with vestibular, and/or ocular motor system assessments. Clinicians should use the VOMS with caution immediately following activity and allow time for recovery from acute fatigue.
Ambient mass spectrometry (MS) has revolutionized the way of MS analysis and broadened its application in various fields. This paper describes the use of microfluidic techniques to simplify the setup ...and improve the functions of ambient MS by integrating the sampling probe, electrospray emitter probe, and online mixer on a single glass microchip. Two types of sampling probes, including a parallel-channel probe and a U-shaped channel probe, were designed for dry-spot and liquid-phase droplet samples, respectively. We demonstrated that the microfabrication techniques not only enhanced the capability of ambient MS methods in analysis of dry-spot samples on various surfaces, but also enabled new applications in the analysis of nanoliter-scale chemical reactions in an array of droplets. The versatility of the microchip-based ambient MS method was demonstrated in multiple different applications including evaluation of residual pesticide on fruit surfaces, sensitive analysis of low-ionizable analytes using postsampling derivatization, and high-throughput screening of Ugi-type multicomponent reactions.
Packaged, transferred, and delivered: A method has been developed that automatically transfers the contents of oil‐encapsulated aqueous plugs to a co‐flowing aqueous stream, which enables ...dilution‐free electrospray ionization (ESI) mass spectrometric analysis for droplet‐based microfluidics.
A limitation of conventional bulk-tissue proteome studies in amyotrophic lateral sclerosis (ALS) is the confounding of motor neuron (MN) signals by admixed non-MN proteins. Here, we leverage laser ...capture microdissection and nanoPOTS single-cell mass spectrometry-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control tissues. In a follow-up analysis, we examine the impact of stratification of MNs based on cytoplasmic transactive response DNA-binding protein 43 (TDP-43)+ inclusion pathology on the profiles of 2,238 proteins. We report extensive overlap in differentially abundant proteins identified in ALS MNs with or without overt TDP-43 pathology, suggesting early and sustained dysregulation of cellular respiration, mRNA splicing, translation, and vesicular transport in ALS. Together, these data provide insights into proteome-level changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein dynamics in human neurologic diseases.
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•Single-cell proteomic analysis of human ALS motor neurons directly captured by LMD•ALS motor neuron deficiencies in splicing, translation, and vesicular transport machinery•Stratification of single-neuron proteomes based on pathologic TDP-43 inclusions•Utility of trace sample proteomics in augmenting the study of human neurological diseases
Guise and Misal et al. report the unbiased single-cell proteomic analysis of ALS motor neurons directly captured from human tissues to explore disease-associated protein dynamics across TDP43-pathological strata. This study highlights both important technical challenges accompanying single-cell omics studies and emerging avenues for exploring human disease biology with nanoPOTS.
Sample preparation for single-cell proteomics is generally performed in a one-pot workflow with multiple dispensing and incubation steps. These hours-long processes can be labor intensive and lead to ...long sample-to-answer times. Here we report a sample preparation method that achieves cell lysis, protein denaturation, and digestion in 1 h using commercially available high-temperature-stabilized proteases with a single reagent dispensing step. Four different one-step reagent compositions were evaluated, and the mixture providing the highest proteome coverage was compared to the previously employed multistep workflow. The one-step preparation increases proteome coverage relative to the previous multistep workflow while minimizing labor input and the possibility of human error. We also compared sample recovery between previously used microfabricated glass nanowell chips and injection-molded polypropylene chips and found the polypropylene provided improved proteome coverage. Combined, the one-step sample preparation and the polypropylene substrates enabled the identification of an average of nearly 2400 proteins per cell using a standard data-dependent workflow with Orbitrap mass spectrometers. These advances greatly simplify sample preparation for single-cell proteomics and broaden accessibility with no compromise in terms of proteome coverage.
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