Graphene sandwich-based biological specimen preparation for cryo-EM analysis.

High-quality specimen preparation plays a crucial role in cryo-electron microscopy (cryo-EM) structural analysis. In this study, we have developed a reliable and convenient technique called the graphene sandwich method for preparing cryo-EM specimens. This method involves using two layers of graphene films that enclose macromolecules on both sides, allowing for an appropriate ice thickness for cryo-EM analysis. The graphene sandwich helps to mitigate beam-induced charging effect and reduce particle motion compared to specimens prepared using the traditional method with graphene support on only one side, therefore improving the cryo-EM data quality. These advancements may open new opportunities to expand the use of graphene in the field of biological electron microscopy.

Current concepts, advances, and challenges in deciphering the human microbiota with metatranscriptomics.

Metatranscriptomics refers to the analysis of the collective microbial transcriptome of a sample. Its increased utilization for the characterization of human-associated microbial communities has enabled the discovery of many disease-state related microbial activities. Here, we review the principles of metatranscriptomics-based analysis of human-associated microbial samples. We describe strengths and weaknesses of popular sample preparation, sequencing, and bioinformatics approaches and summarize strategies for their use. We then discuss how human-associated microbial communities have recently been examined and how their characterization may change. We conclude that metatranscriptomics insights into human microbiotas under health and disease have not only expanded our knowledge on human health, but also opened avenues for rational antimicrobial drug use and disease management.

µPhos: a scalable and sensitive platform for high-dimensional phosphoproteomics.

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos ('microPhos'), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation.

An Automated Nanowell-Array Workflow for Quantitative Multiplexed Single-Cell Proteomics Sample Preparation at High Sensitivity.

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.

HowDirty: An R package to evaluate molecular contaminants in LC-MS experiments.

Contaminants derived from consumables, reagents, and sample handling often negatively affect LC-MS data acquisition. In proteomics experiments, they can markedly reduce identification performance, reproducibility, and quantitative robustness. Here, we introduce a data analysis workflow combining MS1 feature extraction in Skyline with HowDirty, an R-markdown-based tool, that automatically generates an interactive report on the molecular contaminant level in LC-MS data sets. To facilitate the interpretation of the results, the HTML report is self-contained and self-explanatory, including plots that can be easily interpreted. The R package HowDirty is available from https://github.com/DavidGZ1/HowDirty. To demonstrate a showcase scenario for the application of HowDirty, we assessed the impact of ultrafiltration units from different providers on sample purity after filter-assisted sample preparation (FASP) digestion. This allowed us to select the filter units with the lowest contamination risk. Notably, the filter units with the lowest contaminant levels showed higher reproducibility regarding the number of peptides and proteins identified. Overall, HowDirty enables the efficient evaluation of sample quality covering a wide range of common contaminant groups that typically impair LC-MS analyses, facilitating corrective or preventive actions to minimize instrument downtime.

Assessing the precision of a detergent-assisted cartridge precipitation workflow for non-targeted quantitative proteomics.

Detergent-based workflows incorporating sodium dodecyl sulfate (SDS) necessitate additional steps for detergent removal ahead of mass spectrometry (MS). These steps may lead to variable protein recovery, inconsistent enzyme digestion efficiency, and unreliable MS signals. To validate a detergent-based workflow for quantitative proteomics, we herein evaluate the precision of a bottom-up sample preparation strategy incorporating cartridge-based protein precipitation with organic solvent to deplete SDS. The variance of data-independent acquisition (SWATH-MS) data was isolated from sample preparation error by modelling the variance as a function of peptide signal intensity. Our SDS-assisted cartridge workflow yield a coefficient of variance (CV) of 13%-14%. By comparison, conventional (detergent-free) in-solution digestion increased the CV to 50%; in-gel digestion provided lower CVs between 14% and 20%. By filtering peptides predicting to display lower precision, we further enhance the validity of data in global comparative proteomics. These results demonstrate the detergent-based precipitation workflow is a reliable approach for in depth, label-free quantitative proteome analysis.

ChipFilter: Microfluidic-Based Comprehensive Sample Preparation Methodology for Microbial Consortia.

The metaproteomic approach is an attractive way to describe a microbiome at the functional level, allowing the identification and quantification of proteins across a broad dynamic range as well as the detection of post-translational modifications. However, it remains relatively underutilized, mainly due to technical challenges that should be addressed, including the complexity of extracting proteins from heterogeneous microbial communities. Here, we show that a ChipFilter microfluidic device coupled to a liquid chromatography tandem mass spectrometry (LC-MS/MS) setup can be successfully used for the identification of microbial proteins. Using cultures of Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, we have shown that it is possible to directly lyse the cells and digest the proteins in the ChipFilter to allow the identification of a higher number of proteins and peptides than that by standard protocols, even at low cell density. The peptides produced are overall longer after ChipFilter digestion but show no change in their degree of hydrophobicity. Analysis of a more complex mixture of 17 species from the gut microbiome showed that the ChipFilter preparation was able to identify and estimate the amounts of 16 of these species. These results show that ChipFilter can be used for the proteomic study of microbiomes, particularly in the case of a low volume or cell density. The mass spectrometry data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD039581.

Differences in Protein Capture by SP3 and SP4 Demonstrate Mechanistic Insights of Proteomics Cleanup Techniques.

The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identifications. Single-pot, solid-phase-enhanced sample preparation (SP3) is a cleanup technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. Recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer cleanup technique that employs protein aggregation to capture proteins without modified particles. We hypothesize that differences in capture mechanisms of SP3 and SP4 affect which proteins are identified by each cleanup technique. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. Ultimately, we demonstrate that protein-capture mechanisms are distinct, and the selection of a cleanup technique that yields high proteome coverage is dependent on protein-sample hydrophobicity. Data has been deposited into MassIVE (MSV000094130) and ProteomeXchange (PXD049965).

A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics.

Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-dodecyl ß-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3-4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%-10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 µg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.

Microfluidic Impedance Cytometry Enabled One-Step Sample Preparation for Efficient Single-Cell Mass Spectrometry.

Single-cell mass spectrometry (MS) is significant in biochemical analysis and holds great potential in biomedical applications. Efficient sample preparation like sorting (i.e., separating target cells from the mixed population) and desalting (i.e., moving the cells off non-volatile salt solution) is urgently required in single-cell MS. However, traditional sample preparation methods suffer from complicated operation with various apparatus, or insufficient performance. Herein, a one-step sample preparation strategy by leveraging label-free impedance flow cytometry (IFC) based microfluidics is proposed. Specifically, the IFC framework to characterize and sort single-cells is adopted. Simultaneously with sorting, the target cell is transferred from the local high-salinity buffer to the MS-compatible solution. In this way, one-step sorting and desalting are achieved and the collected cells can be directly fed for MS analysis. A high sorting efficiency (>99%), cancer cell purity (≈87%), and desalting efficiency (>99%), and the whole workflow of impedance-based separation and MS analysis of normal cells (MCF-10A) and cancer cells (MDA-MB-468) are verified. As a standalone sample preparation module, the microfluidic chip is compatible with a variety of MS analysis methods, and envisioned to provide a new paradigm in efficient MS sample preparation, and further in multi-modal (i.e., electrical and metabolic) characterization of single-cells.

CryoEM grid preparation: a closer look at advancements and impact of preparation mode and new approaches.

Sample preparation can present a significant hurdle within single particle cryo-electron microscopy (cryoEM), resulting in issues with reproducibility, data quality or an inability to visualise the sample. There are several factors which can influence this, including sample or buffer composition, grid type, route of sample preparation and interactions with the air-water interface (AWI). Here, we review some of the current routes for sample preparation and the associated challenges. We discuss a range of approaches for overcoming these challenges, such as minimising the grid preparation time, surfactants, grid type and biochemical approaches such as nanomagnetic beads. Finally, we discuss how a set of commercially available protein samples may serve as a benchmark suite for future technologies. This provides a route to compare techniques' abilities not just to generate high-resolution structures but also to overcome the challenges traditionally associated with cryoEM. As the field continues to produce new approaches to sample preparation and we start to better understand the underlying principles behind the behaviour of proteins within a thin film and in response to different environments, especially grid composition, it is hoped that more universal solutions can be provided that make the intractable systems tractable, improve resolution and, importantly, speed up data collection and reduce the currently required dataset sizes.

Sample preparation and culture condition effects on MALDI-TOF MS identification of bacteria: A review.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an excellent tool for bacterial identification. It allows high throughput, sensitive and specific applications in clinical diagnostics and environmental research. Currently, there is no optimal standardized protocol for sample preparation and culture conditions to profile bacteria. The performance of MALDI-TOF MS is affected by several variables, such as sample preparation, culture media and culture conditions, incubation time/growth stage, incubation temperature, high salt content, blood in the culture media, and others. This review thus aims to clarify why a uniformed protocol is not plausible, to assess the effects these factors have on MALDI-TOF MS identification score, and discuss possible optimizations for its methodology, in relation to specific bacterial representatives and strain requirements.

Automated proteomic sample preparation: The key component for high throughput and quantitative mass spectrometry analysis.

Sample preparation for mass spectrometry-based proteomics has many tedious and time-consuming steps that can introduce analytical errors. In particular, the steps around the proteolytic digestion of protein samples are prone to inconsistency. One route for reliable sample processing is the development and optimization of a workflow utilizing an automated liquid handling workstation. Diligent assessment of the sample type, protocol design, reagents, and incubation conditions can significantly improve the speed and consistency of preparation. When combining robust liquid chromatography-mass spectrometry with either discovery or targeted methods, automated sample preparation facilitates increased throughput and reproducible quantitation of biomarker candidates. These improvements in analysis are also essential to process the large patient cohorts necessary to validate a candidate biomarker for potential clinical use. This article reviews the steps in the workflow, optimization strategies, and known applications in clinical, pharmaceutical, and research fields that demonstrate the broad utility for improved automation of sample preparation in the proteomic field.

Analysis of vitamin D metabolic markers by mass spectrometry: Recent progress regarding the "gold standard" method and integration into clinical practice.

Liquid chromatography/tandem mass spectrometry is firmly established today as the gold standard technique for analysis of vitamin D, both for vitamin D status assessments as well as for measuring complex and intricate vitamin D metabolic fingerprints. While the actual mass spectrometry technology has seen only incremental performance increases in recent years, there have been major, very impactful changes in the front- and back-end of MS-based vitamin D assays; for example, the extension to new types of biological sample matrices analyzed for an increasing number of different vitamin D metabolites, novel sample preparation techniques, new powerful chemical derivatization reagents, as well the continued integration of high resolution mass spectrometers into clinical laboratories, replacing established triple-quadrupole instruments. At the same time, the sustainability of mass spectrometry operation in the vitamin D field is now firmly established through proven analytical harmonization and standardization programs. The present review summarizes the most important of these recent developments.

Spatio-temporal plant hormonomics: From tissue to subcellular resolution.

Due to technological advances in mass spectrometry, significant progress has been achieved recently in plant hormone research. Nowadays, plant hormonomics is well established as a fully integrated scientific field focused on the analysis of phytohormones, mainly on their isolation, identification and spatiotemporal quantification in plants. This review represents a comprehensive meta-study of the advances in the phytohormone analysis by mass spectrometry over the past decade. To address current trends and future perspectives, Web of Science data were systematically collected and key features such as mass spectrometry-based analyses were evaluated using multivariate data analysis methods. Our findings showed that plant hormonomics is currently divided into targeted and untargeted approaches. Both aim to miniaturize the sample, allowing high-resolution quantification to be covered in plant organs as well as subcellular compartments. Therefore, we can study plant hormone biosynthesis, metabolism and signalling at a spatio-temporal resolution. Moreover, this trend has recently been accelerated by technological advances such as fluorescence-activated cell sorting or mass spectrometry imaging.

Comparisons of different extraction methods and solvents for saliva samples.

INTRODUCTION: Changes in the categories and concentrations of salivary metabolites may be closely related to oral, intestinal or systemic diseases. To study salivary metabolites, the first analytical step is to extract them from saliva samples as much as possible, while reducing interferences to a minimum. Frequently used extraction methods are protein precipitation (PPT), liquid-liquid extraction (LLE) and solid-phase extraction (SPE), with various organic solvents. The types and quantities of metabolites extracted with different methods may vary greatly, but few studies have systematically evaluated them. OBJECTIVES: This study aimed to select the most suitable methods and solvents for the extraction of saliva according to different analytical targets. METHODS: An untargeted metabolomics approach based on liquid chromatography-mass spectrometry was applied to obtain the raw data. The numbers of metabolites, repeatability of the data and intensities of mass spectrometry signals were used as evaluation criteria. RESULTS: PPT resulted in the highest coverage. Among the PPT solvents, acetonitrile displayed the best repeatability and the highest coverage, while acetone resulted in the best signal intensities for the extracted compounds. LLE with the mixture of chloroform and methanol was the most suitable for the extraction of small hydrophobic compounds. CONCLUSION: PPT with acetonitrile or acetone was recommended for untargeted analysis, while LLE with the mixture of chloroform and methanol was recommended for small hydrophobic compounds.

Automated sample preparation for electrospray ionization mass spectrometry based on CLOCK-controlled autonomous centrifugal microfluidics.

We report a centrifugal microfluidic device that automatically performs sample preparation under steady-state rotation for clinical applications using mass spectrometry. The autonomous microfluidic device was designed for the control of liquid operation on centrifugal hydrokinetics (CLOCK) paradigm. The reported device was highly stable, with less than 7% variation with respect to the time of each unit operation (sample extraction, mixing, and supernatant extraction) in the preparation process. An agitation mechanism with bubbling was used to mix the sample and organic solvent in this device. We confirmed that the device effectively removed the protein aggregates from the sample, and the performance was comparable to those of conventional manual sample preparation procedures that use high-speed centrifugation. In addition, probe electrospray ionization mass spectrometry (PESI-MS) was performed to compare the device-treated and manually treated samples. The obtained PESI-MS spectra were analyzed by partial least squares discriminant analysis, and the preparation capability of the device was found to be equivalent to that of the conventional method.

Studying crystallisation processes using electron microscopy: The importance of sample preparation.

We present a comparison of common electron microscopy sample preparation methods for studying crystallisation processes from solution using both scanning and transmission electron microscopy (SEM and TEM). We focus on two widely studied inorganic systems: calcium sulphate, gypsum (CaSO4·2H2O) and calcium carbonate (CaCO3). We find significant differences in crystallisation kinetics and polymorph selection between the different sample preparation methods, which indicate that drying and chemical quenching can induce severe artefacts that are capable of masking the true native state of the crystallising solution. Overall, these results highlight the importance of cryogenic (cryo)-quenching crystallising solutions and the use of full cryo-TEM as the most reliable method for studying the early stages of crystallisation.

Quantification of surface grinding during the sample preparation of cementitious materials by optical profilometry.

Sample preparation is of utmost importance for any microscopy and microstructural analysis. Correct preparation will allow accurate interpretation of microstructural features. A well-polished section is essential when scanning electron microscopy (SEM) is used in backscattering electron (BSE) mode and characteristic X-rays are to be quantified using an energy-dispersive spectroscopy (EDS) detector. However, obtaining a well-polished section, especially for cementitious materials containing aggregates, is considered to be challenging and requires experience. A sample preparation procedure consists of cutting, grinding and polishing. Undercutting of soft and brittle paste between harder aggregates can be overcome by vacuum epoxy impregnation offering mechanical support in the matrix. Furthermore, most of the attention during the sample preparation is given to the polishing of the sample. There is a wide range of suggestions on polishing steps, ranging from grain sizes, time and applied force; however, the final assessment of a polish surface is often subjective and qualitative. Therefore, a quantitative, reproducible guidance on the grinding steps, effect of experimental parameters and the influence of different grinding steps on the surface quality are required. In this paper, the influence of grinding was quantitatively evaluated by a digital microscope equipped with optical profilometry tools, through a step-wise procedure, including sample orientation, grinding time and the difference between cement paste and concrete. Throughout the grinding procedure, the surface profiles were determined after each grinding step. This showed the step-wise change in surface roughness and quality during the grinding procedure. Finally, the surface qualities were evaluated using optical and electron microscopy, which show the importance of the grinding/prepolishing steps during sample preparation.

Determination of biomarkers of chlorine exposure from biological samples: a review of analysis techniques.

Introduction: Chlorine gas can be toxic when inhaled or absorbed at high concentrations through the skin. It can cause pulmonary edema, pulmonary inflammation, respiratory failure, and potentially death. Monitoring chlorine exposure helps in determining treatment regimens and may inform safeguards, such as personal protective equipment and ventilation systems. Therefore, verification of chlorine exposure is crucial to protecting human health. This has led to identification of multiple biomarkers of Cl2 exposure with associated innovations in methods of analysis to monitor these markers.Materials and methods: In this review of the last 30 years of literature, biomarkers and associated methods of detection for the determination of chlorine exposure from biological samples are detailed and critically evaluated.Results and discussion: From the 36 included studies, the most useful biomarkers for Cl2 exposure include tyrosine adducts, chlorohydrin, chloro-fatty-acids, chloro-fatty-aldehydes, and chloro-fatty-alcohols. The most common sample preparation methods for these markers are hydrolysis and extraction and the most common analysis techniques are chromatographic separation with mass spectrometric detection.Conclusion: The findings of this review emphasize the need for continued research into biomarkers and stronger evaluation of proposed analytical methods, including validation, to allow more appropriate comparison, which will ultimately improve patient outcomes.