A rapid and online microvolume flow-through dialysis probe designed for sample preparation in the analysis of veterinary drug residues is introduced. This study addresses the need for efficient and green sample preparation methods that reduce chemical waste and reagent use. The dialysis probe integrates with liquid chromatography and mass spectrometry (LC-MS) systems, facilitating automated, high-throughput analysis. The dialysis method utilizes minimal reagent volumes per sample, significantly reducing the generation of solvent waste compared to traditional sample preparation techniques. Several veterinary drugs were spiked into tissue homogenates and analyzed to validate the probe\'s efficacy. A diagnostic sensitivity of >97% and specificity of >95% were obtained for this performance evaluation. The results demonstrated the effective removal of cellular debris and particulates, ensuring sample integrity and preventing instrument clogging. The automated dialysis probe yielded recovery rates between 27 and 77% for multiple analytes, confirming its potential to streamline veterinary drug residue analysis, while adhering to green chemistry principles. The approach highlights substantial improvements in both environmental impact and operational efficiency, presenting a viable alternative to conventional sample preparation methods in regulatory and research applications.

Patulin, a toxic mycotoxin, can contaminate apple-derived products. The FDA has established an action level of 50 ppb (ng/g) for patulin in apple juice and apple juice products. To effectively monitor this mycotoxin, there is a need for adequate analytical methods that can reliably and efficiently determine patulin levels. In this work, we developed an automated sample preparation workflow followed by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) detection to identify and quantify patulin in a single method, further expanding testing capabilities for monitoring patulin in foods compared to traditional optical methods. Using a robotic sample preparation system, apple juice, apple cider, apple puree, apple-based baby food, applesauce, fruit rolls, and fruit jam were fortified with 13C-patulin and extracted using dichloromethane (DCM) without human intervention, followed by an LC-APCI-MS/MS analysis in negative ionization mode. The method achieved a limit of quantification of 4.0 ng/g and linearity ranging from 2 to 1000 ng/mL (r2 > 0.99). Quantitation was performed with isotope dilution using 13C-patulin as an internal standard and solvent calibration standards. Average recoveries (relative standard deviations, RSD%) in seven spike matrices were 95% (9%) at 10 ng/g, 110% (5%) at 50 ng/g, 101% (7%) at 200 ng/g, and 104% (4%) at 1000 ng/g (n = 28). The ranges of within-matrix and between-matrix variability (RSD) were 3-8% and 4-9%, respectively. In incurred samples, the identity of patulin was further confirmed with a comparison of the information-dependent acquisition-enhanced product ion (IDA-EPI) MS/MS spectra to a reference standard. The metrological traceability of the patulin measurements in an incurred apple cider (21.1 ± 8.0 µg/g) and apple juice concentrate (56.6 ± 15.6 µg/g) was established using a certified reference material and calibration data to demonstrate data confidence intervals (k = 2, 95% confidence interval).

Membrane proteins are underrepresented during proteome characterizations, primarily owing to their lower solubility. Sodium dodecyl sulfate (SDS) is favored to enhance protein solubility but interferes with downstream analysis by mass spectrometry. Here, we present an improved workflow for SDS depletion using transmembrane electrophoresis (TME) while retaining a higher recovery of membrane proteins. Though higher levels of organic solvent lower proteome solubility, we found that the inclusion of 40% methanol provided optimal solubility of membrane proteins, with 86% recovery relative to extraction with SDS. Incorporating 40% methanol during the electrophoretic depletion of SDS by TME also maximized membrane protein recovery. We further report that methanol accelerates the rate of detergent removal, allowing TME to deplete SDS below 100 ppm in under 3 min. This is attributed to a three-fold elevation in the critical micelle concentration (CMC) of SDS in the presence of methanol, combined with a reduction in the SDS to protein binding ratio in methanol (0.3 g SDS/g protein). MS analysis of membrane proteins isolated from the methanol-assisted workflow revealed enhanced proteome detection, particularly for proteins whose pI contributed a minimal net charge and therefore possessed reduced solubility in a purely aqueous solvent. This protocol presents a robust approach for the preparation of membrane proteins by maximizing their solubility in MS-compatible solvents, offering a tool to advance membrane proteome characterization.

Generic extraction methods for the multi-compound pesticide analysis of food have found their solid place in laboratories. Ethyl acetate and acetonitrile extraction methods have been developed as fast and easy to handle standard multi-compound methods, both feature benefits and limitations. The direct injection to gas chromatography can be impaired by a high burden of coextracted matrix, resulting in deterioration of the chromatographic system and matrix effects, requiring frequent maintenance. Therefore, common clean-up methods, such as dispersive solid-phase extraction, freeze-out of fats, or gel permeation chromatography, have been applied in clean-up. Automated clean-up using micro-solid-phase extraction (µSPE) is a recent development with several demonstrated advantages when employed in the analysis of pesticides and other contaminants in foods extracted with acetonitrile, but it has not yet been evaluated in this application using ethyl acetate for extraction. In this study, an automated procedure using µSPE cartridges was developed and established on an x,y,z robotic sampler for the raw extract clean-up and preparation of diluted samples for injection on a GC-MS/MS system. Validation experiments for 212 pesticides, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons in lettuce, avocado, raspberry, paprika, egg, and liver extracts were performed using µSPE with MgSO4, PSA, C18, and CarbonX. The performance in routine operation is briefly discussed.

Urine provides a diverse source of information related to a patient\'s health status and is ideal for clinical proteomics due to its ease of collection. To date, most methods for the preparation of urine samples lack the throughput required to analyze large clinical cohorts. To this end, we developed a novel workflow, urine-HILIC (uHLC), based on an on-bead protein capture, clean-up, and digestion without the need for bottleneck processing steps such as protein precipitation or centrifugation. The workflow was applied to an acute kidney injury (AKI) pilot study. Urine from clinical samples and a pooled sample was subjected to automated sample preparation in a KingFisher™ Flex magnetic handling station using the novel approach based on MagReSyn® HILIC microspheres. For benchmarking, the pooled sample was also prepared using a published protocol based on an on-membrane (OM) protein capture and digestion workflow. Peptides were analyzed by LCMS in data-independent acquisition (DIA) mode using a Dionex Ultimate 3000 UPLC coupled to a Sciex 5600 mass spectrometer. The data were searched in Spectronaut™ 17. Both workflows showed similar peptide and protein identifications in the pooled sample. The uHLC workflow was easier to set up and complete, having less hands-on time than the OM method, with fewer manual processing steps. Lower peptide and protein coefficient of variation was observed in the uHLC technical replicates. Following statistical analysis, candidate protein markers were filtered, at ≥8.35-fold change in abundance, ≥2 unique peptides and ≤1% false discovery rate, and revealed 121 significant, differentially abundant proteins, some of which have known associations with kidney injury. The pilot data derived using this novel workflow provide information on the urinary proteome of patients with AKI. Further exploration in a larger cohort using this novel high-throughput method is warranted.

Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lags. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation including multiplexed labeling up to 16-plex with high sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE, reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1500 protein groups per TMT10-plex with reduced or eliminated need for a carrier proteome. We evaluated the effect of wider precursor isolation windows at single-cell input levels and found that using 2 Da isolation windows increased overall sensitivity without significantly impacting interference. Using the dedicated mass spectrometry acquisition strategies detailed here, we identified on average close to 2000 proteins per TMT10-plex across 170 multiplexed single cells that readily distinguished human cell types. Overall, our workflow combines highly efficient sample preparation, chromatographic and ion mobility-based filtering, rapid wide-window data-dependent acquisition analysis, and intelligent data analysis for optimal multiplexed single-cell proteomics. This versatile and automated proteoCHIP-based sample preparation approach is sufficiently sensitive to drive biological applications of single-cell proteomics and can be readily adopted by proteomics laboratories.

Biological testing on the International Space Station (ISS) is necessary in order to monitor the microbial burden and identify risks to crew health. With support from a NASA Phase I Small Business Innovative Research contract, we have developed a compact prototype of a microgravity-compatible, automated versatile sample preparation platform (VSPP). The VSPP was built by modifying entry-level 3D printers that cost USD 200-USD 800. In addition, 3D printing was also used to prototype microgravity-compatible reagent wells and cartridges. The VSPP\'s primary function would enable NASA to rapidly identify microorganisms that could affect crew safety. It has the potential to process samples from various sample matrices (swab, potable water, blood, urine, etc.), thus yielding high-quality nucleic acids for downstream molecular detection and identification in a closed-cartridge system. When fully developed and validated in microgravity environments, this highly automated system will allow labor-intensive and time-consuming processes to be carried out via a turnkey, closed system using prefilled cartridges and magnetic particle-based chemistries. This manuscript demonstrates that the VSPP can extract high-quality nucleic acids from urine (Zika viral RNA) and whole blood (human RNase P gene) in a ground-level laboratory setting using nucleic acid-binding magnetic particles. The viral RNA detection data showed that the VSPP can process contrived urine samples at clinically relevant levels (as low as 50 PFU/extraction). The extraction of human DNA from eight replicate samples showed that the DNA extraction yield is highly consistent (there was a standard deviation of 0.4 threshold cycle when the extracted and purified DNA was tested via real-time polymerase chain reaction). Additionally, the VSPP underwent 2.1 s drop tower microgravity tests to determine if its components are compatible for use in microgravity. Our findings will aid future research in adapting extraction well geometry for 1 g and low g working environments operated by the VSPP. Future microgravity testing of the VSPP in the parabolic flights and in the ISS is planned.

Vitamin D belongs to the fat-soluble vitamins and is an integral part of bone metabolism. In the human body, a decreased vitamin D level can be an additional risk factor for diseases like cancer, diabetes, and mental diseases. As a result, an enormous increase in the demand for vitamin D testing has been observed in recent years, increasing the demand for powerful methods for vitamin D determination at the same time.Automation is the key factor in increasing sample throughput. This study compares three fully automated sample preparation methods for the determination of 25(OH)D2 and 25(OH)D3 in plasma and serum samples. Starting from a semiautomated reference method, the method is tested manually and subsequently fully automated on the Biomek i7 Workstation by integrating a centrifuge and a positive pressure extractor into the workstation. Alternatively, the centrifugation for the separation of protein aggregates and supernatant is replaced by a filter plate. Finally, the sample throughput is further increased by using phospholipid removal cartridges. The results show that phospholipid removal significantly increases the recovery rates in liquid chromatography-mass spectrometry. With the phospholipid removal cartridges, recovery rates of 97.36% for 25(OH)D2 and 102.5% for 25(OH)D3 were achieved, whereas with the automated classic automated preparation method, the recovery rates were 83.31% for 25(OH)D2 and 86.54% for 25(OH)D3. In addition to the technical evaluation, the different methods were also examined with regard to their economic efficiency. Finally, the qualitative and quantitative performance of the developed methods is benchmarked with a selected semiautomatic reference method.

The present work aims to a promising re-utilization of the massive waste derived from the tuna fishing industry, for which by-products can represent more than 50% of the original material. Due to the considerable content in polyunsaturated fatty acids and noble proteins, such wastes can be used as primary source of functional ingredients in the production of nutraceuticals. The composition of the lipid and protein tuna fractions was investigated by means of gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry methods (in wastes and edible parts), and a preliminary characterization of potential bioactive peptides was achieved. Automated sample preparation allowed speeding up the analytical workflow, while allowing for highly sensitive and selective lipid characterization. The ω3 fatty acid content was found higher in waste products compared to the muscle, in terms of fatty acids as well as complex lipids. As for peptides, extraction by isoelectric solubilization/precipitation was performed, followed by enzymatic digestion and high-performance liquid chromatography-tandem mass spectrometry analysis. Furthermore, the use of bioinformatics tools highlighted the presence of potential antimicrobial peptides in the samples investigated.

Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial information in situ at single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, non-adherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines, peripheral blood mononucleated cells (PBMC) and human platelets on an adherent surface. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.