Sample Preparation Protocol Research Articles

Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead–based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (μg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 μg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 μg of beads for sample preparation and long-term digestion (16h) with 0.15 μg Lys-C and 2.5 μg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.

An Automated and Fast Sample Preparation Workflow for Laser Microdissection Guided Ultrasensitive Proteomics

Spatial tissue proteomics integrating whole-slide imaging, laser microdissection, and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE robotic system, which has the capacity to process 192 samples in 3 h. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking, and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows ‘on-the-fly’ sample clean-up, particularly pertinent for laser microdissection workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell, and epithelial microregions of 4000 μm2 to a depth of ∼2000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.

Protocols for preparation of biomineralized samples for computed tomography

Computed tomography spans a versatile set of techniques that range several length scales and modalities. It is common to them all that the sample must be prepared in a way which allows for addressing the scientific question(s) posed as well as being suitable for the specific setup of the experiment. We present two lathe-based sample preparation workflows developed to prepare biomineralized samples (here bone) for two very different experiments in terms of setup, types of questions asked, and sample requirements. The first experiment, involving the measurement of high throughput (synchrotron) micro-computed tomography, required the preparation of many samples with homogeneity in size, shape, and bone site. This was achieved through a particular sequence of cutting and embedding steps finalized by lathe milling. The resulting samples were cylindrical in shape with diameters close to the field of view of the ensuing tomography experiment, which allowed maximizing the investigated sample volumes. The second experiment was a combined ptychography and X-ray fluorescence nano-computed tomography experiment, which required preparation of a few-micrometer-sized sample. Moreover, the scientific interest was in a specific, localized feature in bone. Thus, the sample had to be extracted from a precise location from within the whole bone. Again, the developed workflow comprised many steps, including both lathe milling and focused ion beam milling. Importantly, localized preparation was enabled by measuring in-house X-ray micro-computed tomography at crucial points in the workflow. The presented workflows provide examples of preparation pathways that can be standardized and strongly increase the throughput, quality, and success rate of tomography experiments.

Heat 'n Beat: A Universal High-Throughput End-to-End Proteomics Sample Processing Platform in under an Hour.

Proteomic analysis by mass spectrometry of small (≤2 mg) solid tissue samples from diverse formats requires high throughput and comprehensive proteome coverage. We developed a nearly universal, rapid, and robust protocol for sample preparation, suitable for high-throughput projects that encompass most cell or tissue types. This end-to-end workflow extends from original sample to loading the mass spectrometer and is centered on a one-tube homogenization and digestion method called Heat 'n Beat (HnB). It is applicable to most tissues, regardless of how they were fixed or embedded. Sample preparation was divided into separate challenges. The initial sample washing and final peptide cleanup steps were adapted to three tissue sources: fresh frozen (FF), optimal cutting temperature (OCT) compound embedded (FF-OCT), and formalin-fixed paraffin embedded (FFPE). Third, for core processing, tissue disruption and lysis were decreased to a 7 min heat and homogenization treatment, and reduction, alkylation, and proteolysis were optimized into a single step. The refinements produced near doubled peptide yield when compared to our earlier method ABLE delivered a consistently high digestion efficiency of 85-90%, reported by ProteinPilot, and required only 38 min for core processing in a single tube, with the total processing time being 53-63 min. The robustness of HnB was demonstrated on six organ types, a cell line, and a cancer biopsy. Its suitability for high-throughput applications was demonstrated on a set of 1171 FF-OCT human cancer biopsies, which were processed for end-to-end completion in 92 h, producing highly consistent peptide yield and quality for over 3513 MS runs.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of phenobarbital in human serum and plasma.

Phenobarbital serves as an antiepileptic drug (AED) and finds application in the treatment of epilepsy either as monotherapy or adjunctive therapy. This drug exhibits various pharmacodynamic properties that account for its beneficial effects as well as potential side effects. Accurate measurement of its concentration is critical for optimizing AED therapy through appropriate dose adjustments. Therefore, our objective was to develop and validate a new reference measurement procedure (RMP) for the accurate quantification of phenobarbital levels in human serum and plasma. A sample preparation protocol based on protein precipitation followed by a high dilution step was established in combination with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a C8 column to separate target analytes from known and unknown interferences. Assay validation and determination of measurement uncertainty were performed based on current guidelines. Selectivity and Specificity were assessed using spiked serum and plasma samples; to investigate possible matrix effects (MEs) a post-column infusion experiment and a comparison of standard line slopes was performed. Precision and accuracy were determined within a multiday precision experiment. The RMP was shown to be highly selective and specific, with no evidence of matrix interferences. It can be used to quantify phenobarbital in the range of 1.92 to 72.0 μg/mL. Intermediate precision was less than 3.2 %, and repeatability coefficient of variation (CV) ranged from 1.3 to 2.0 % across all concentration levels. The relative mean bias ranged from -3.0 to -0.7 % for native serum levels, and from -2.8 to 0.8 % for Li-heparin plasma levels. The measurement uncertainties (k=1) for single measurements and target value assignment were 1.9 to 3.3 % and 0.9 to 1.6 %, respectively. A novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma is presented which can be used for the standardization of routine assays and the evaluation of clinically relevant samples.

Elastic properties and tensile strength of 2D Ti3C2Tx MXene monolayers

Two-dimensional (2D) transition metal nitrides and carbides (MXenes), represented by Ti3C2Tx, have broad applications in flexible electronics, electromechanical devices, and structural membranes due to their unique physical and chemical properties. Despite the Young’s modulus of 2D Ti3C2Tx has been theoretically predicted to be 0.502 TPa, which has not been experimentally confirmed so far due to the measurement is extremely restricted. Here, by optimizing the sample preparation, cutting, and transfer protocols, we perform the direct in-situ tensile tests on monolayer Ti3C2Tx nanosheets using nanomechanical push-to-pull equipment under a scanning electron microscope. The effective Young’s modulus is 0.484 ± 0.013 TPa, which is much closer to the theoretical value of 0.502 TPa than the previously reported 0.33 TPa by the disputed nanoindentation method, and the measured elastic stiffness is ~948 N/m. Moreover, during the process of tensile loading, the monolayer Ti3C2Tx shows an average elastic strain of ~3.2% and a tensile strength as large as ~15.4 GPa. This work corrects the previous reports by nanoindentation method and demonstrates that the Ti3C2Tx indeed keeps immense potential for broad range of applications.

A Comparison of Bottom-Up Proteomic Sample Preparation Methods for the Human Parasite Trichomonas vaginalis.

Bottom-up proteomic approaches depend on the efficient digestion of proteins into peptides for mass spectrometric analysis. Sample preparation strategies, based on magnetic beads, filter-aided systems, or in-solution digests, are commonly used for proteomic analysis. Time-intensive methods like filter-aided sample preparation (FASP) have led to the development of new, more time-efficient filter-based strategies like suspension trappings (S-Traps) or magnetic bead-based strategies like SP3. S-Traps have been reported as an alternative proteomic sample preparation method as they allow for high sodium dodecyl sulfate (SDS) concentrations to be present in the sample. In this study, we compare the efficiency of different protocols for FASP, SP3, and S-Trap-based digestion of proteins after extraction from Trichomonas vaginalis. Overall, we found a high number of protein IDs for all tested methods and a high degree of reproducibility within each method type. However, FASP with a 3 kDa cutoff filter unit outperformed the other methods analyzed, referring to the number of protein IDs. This is the first work providing the direct comparison of four different bottom-up proteomic approaches regarding the most efficient proteomic sample preparation protocol for the human parasite T. vaginalis.

Successive Protein Extraction Using Hydroxylamine to Increase the Depth of Proteome Coverage in Fresh, Forensic, and Archaeological Bones.

Proteomics is continually being applied to a wider range of applications, now including the analysis of archaeological samples and anatomical specimens, particularly collagen-containing tissues such as bones and teeth. Here, we present the application of a chemical digestion-based proteomics sample preparation protocol to the analysis of fresh, anatomical, and archaeological samples. We describe and discuss two protocols: one that uses hydroxylamine as an additional step of the proteomic workflow, applied to the insoluble fraction, and another that applies hydroxylamine directly on demineralized bones and teeth. We demonstrate the additional information that can be extracted using both protocols, including an increase in the sequence coverage and number of peptides detected in modern and archaeological samples and an increase in the number of proteins identified in archaeological samples. By targeting research related to collagens or extracellular matrix proteins, the use of this protocol will open new insights, considering both fresh and ancient mineralized samples.

Easy Access to Super-Resolution Applications with DeepSIM

Abstract Super-resolution microscopy is becoming more and more relevant and widespread for solving complex biological questions. However, many super-resolution techniques require high-level expertise and specific sample preparation protocols limiting the kind of fluorophore that can be used and consequently the applications. CrestOptics DeepSIM supports researchers deploying standard sample preparation protocols used for confocal microscopy while offering staggering resolution, the possibility of doing live cell imaging, and the ability to image deep into tissues. DeepSIM also allows staining with commonly used fluorescent markers, making it compatible with a large variety of samples and applications.

Net-Shaped DNA Nanostructure-Based Lateral Flow Assays for Rapid and Sensitive SARS-CoV-2 Detection.

Lateral flow assay (LFA)-based rapid antigen tests are experiencing extensive global uptake as an expeditious and highly effective modality for the screening of viral infections during the COVID-19 pandemic. While these devices have played a significant role in alleviating the burden on the public healthcare system, their specificity and sensitivity fall short compared with molecular tests. In this study, we endeavor to address both limitations through the utilization of DNA nanotechnology in LFA format, wherein we substitute the target-specific antibody with designer DNA nanostructure-based molecular probes for recognizing the SARS-CoV-2 virus via multivalent, pattern-matching interactions. We meticulously designed a Net-shaped DNA nanostructure and strategically arranged trimeric clusters of aptamers that specifically recognize the spike proteins of SARS-CoV-2. This approach has proven instrumental in bolstering virus-binding affinity on the LFAs. Our findings indicate high LFA sensitivity, enabling the detection of viral loads ranging from 103 to 108 viral copies/mL. This notable sensitivity is maintained across various SARS-CoV-2 viral strains, obviating the need for intricate sample preparation protocols. The significance of this heightened sensitivity lies in the crucial role played by the designer DNA nanostructure, which facilitates the detection of extremely low levels of viral loads. This not only enhances the overall reliability of self-testing but also reduces the likelihood of false-negative results, especially in cases of low viral load within patient samples.

Salicylic acid as a switchable hydrophilicity solvent for the microextraction of the antibiotic amphotericin B from human urine followed by HPLC-UV analysis

Monitoring the concentration levels of amphotericin B in the human organism is of utmost importance to evaluate the efficacy of the therapy. However, the reported sample preparation protocols for the extraction of this compound are very limited. In this work, salicylic acid is utilized as a switchable hydrophilicity solvent for the microextraction of amphotericin B from human urine samples before the HPLC analysis. The method is based on the phase transition of salicylic acid by changing the pH of the sample enabling efficient dispersion and phase separation in one step. A high contact area of the solidified solvent with the sample solution provided effective extraction of the target analyte. Among others, critical experimental parameters affecting the extraction performance of the drug were systematically investigated. The separation of the drug was carried out on a C8 stationary phase using mixtures of citric acid and methanol as mobile phases. Excellent linearity was observed in the range of 0.1 – 2.0 μg mL−1, while the precision (%RSD) was less than 14.5 % in all cases. The lower limit of quantification was 0.1 μg mL−1, and the limit of detection was 0.03 μg mL−1. The intra-day and inter-day accuracy were satisfactory, being in the range of 85.2 – 109.1 %. The robustness of the microextraction protocol was evaluated using DoE. The green potential and the practicality of the proposed analytical scheme were examined based on Green Analytical Procedure Index (GAPI) and Blue Applicability Grade Index (BAGI), respectively. A BAGI score of 65 was attained, demonstrating good applicability in terms of practicality, while the novel protocol was found to show green merits. The developed method can be used for the analysis of the drug in human urine.

Live cell fluorescence microscopy-an end-to-end workflow for high-throughput image and data analysis.

Fluorescence microscopy images of biological samples contain valuable information but require rigorous analysis for accurate and reliable determination of changes in protein localization, fluorescence intensity, and morphology of the studied objects. Traditionally, cells for microscopy are immobilized using chemicals, which can introduce stress. Analysis often focuses only on colocalization and involves manual segmentation and measurement, which are time-consuming and can introduce bias. Our new workflow addresses these issues by gently immobilizing cells using a small agarose block on a microscope coverslip. This approach is suitable for cell-walled cells (yeast, fungi, plants, bacteria), facilitates their live imaging under conditions close to their natural environment and enables the addition of chemicals during time-lapse experiments. The primary focus of the protocol is on the presented analysis workflow, which is applicable to virtually any cell type-we describe cell segmentation using the Cellpose software followed by automated analysis of a multitude of parameters using custom-written Fiji (ImageJ) macros. The results can be easily processed using the provided R markdown scripts or available graphing software. Our method facilitates unbiased batch analysis of large datasets, improving the efficiency and accuracy of fluorescence microscopy research. The reported sample preparation protocol and Fiji macros were used in our recent publications: Microbiol Spectr (2022), DOI: 10.1128/spectrum.01961-22; Microbiol Spectr (2022), DOI: 10.1128/spectrum.02489-22; J Cell Sci (2023), DOI: 10.1242/jcs.260554.

Quantification of prohibited substances and endogenous corticosteroids in saliva using traditional, alternative microextraction-based, and novel 3D printed sample-preparation methods coupled with LC-MS

Oral fluid has gained significant interest as an alternative matrix for drug testing due to its easy and non-invasive collection. Despite these advantages, achieving suitably low limits of detection remains a clear challenge in the use of oral fluids for drug screening. In this study, we demonstrate that the application of commercially available SPME fibers followed by liquid chromatography tandem mass spectrometry can enable the comprehensive detection and confirmation of drugs in oral fluid samples. To this end, we develop and test a sample-preparation protocol for a panel of 46 drugs covering the most popular drugs of abuse and doping agents available worldwide. Human saliva samples were collected using a Salivette® device (CE IVD certified) and sampled using SPME devices coated with a C18 extraction phase. The proposed protocol was validated with respect to its lower limits of quantification (LLOQ), linearity, matrix effects, precision, and extraction recovery. Linearity was confirmed for all compounds (R2 > 0.97), except for testosterone (R2 = 0.953) and metandrostenolon (R2 = 0.958). Furthermore, 4 compounds suffered from matrix effects, with less than 10 % deviation from acceptance criteria. After analytical validation, saliva samples from volunteers were analyzed to determine free concentrations of cortisol at different times after awaking. Finally, a 3D-printed prototype device was designed and successfully applied to extract small molecules, thus demonstrating a new modern low-cost approach for bioanalysis.

Enhancing affinity purification of monoclonal antibodies from human serum for subsequent CZE-MS analysis

Due to the separation technique employed, capillary electrophoresis coupled to mass spectrometry (CE-MS) analysis performances are significantly influenced by the chemical composition and the complexity of the sample. In various applications, that impact has prevented the use of CE-MS for the characterization and quantification of proteins in biological samples. Here we present the development and evaluation and a sample preparation procedure, based on affinity purification, for the specific extraction of the monoclonal antibody (mAbs) infliximab from human serum in order to perform subsequent proteolytic digestion and CE-MS/MS analysis. Three distinctive sample preparation strategies were envisaged. In each case, the different steps composing the protocol were thoroughly optimized and evaluated in order to provide a sample preparation addressing the important complexity of serums samples while providing an optimal compatibility with CE-MS/MS analysis. The different sample preparation strategies were assessed concerning the possibility to achieve an appropriate absolute quantification of the mAbs using CE-MS/MS for samples mimicking patient serum samples. Also, the possibility to perform the characterization of several types of post-translational modifications (PTMs) was evaluated. The sample preparation protocols allowed the quantification of the mAbs in serums samples for concentration as low as 0.2 µg·mL−1 (2.03 nM) using CE-MS/MS analysis, also the possibility to characterize and estimate the modification level of PTMs hotspots in a consistent manner. Results allowed to attribute the effect on the electrophoretic separation of the different steps composing sample preparation. Finally, they demonstrated that sample preparation for CE-MS/MS analysis could benefit greatly for the extended applicability of this type of analysis for complex biological matrices.

Label-Free Sample Preparation for Single-Cell Proteomics.

In recent years, single-cell proteomics (SCP) has become a valuable addition to other single-cell omics technologies for studying cellular heterogeneity. The amount of protein in a single cell is very limited, and in contrast to sequencing techniques, there are currently no means for protein amplification. Therefore, most single-cell proteomics approaches aim to maximize sample preparation efficiency while minimizing peptide loss. By reducing processing volumes to sub-microliters and avoiding manual transfer steps that could lead to peptide loss, peptide recovery, and the robustness of SCP workflows have been significantly improved. In this chapter, we describe a protocol for label-free SCP sample preparation using the cellenONE® platform and the proteoCHIP LF 48 substrate prior to analysis with high-performance liquid chromatography-mass spectrometry.

Biomedical SERS - the current state and future trends.

Surface enhanced Raman spectroscopy (SERS) is meeting the requirements in biomedical science being a highly sensitive and specific analytical tool. By employing portable Raman systems in combination with customized sample pre-treatment, point-of-care-testing (POCT) becomes feasible. Powerful SERS-active sensing surfaces with high stability and modification layers if required are available for testing and application in complex biological matrices such as body fluids, cells or tissues. This review summarizes the current state in sample collection and pretreatment in SERS detection protocols, SERS detection schemes, i.e. direct and indirect SERS as well as targeted and non-targeted SERS, and SERS-active sensing surfaces. Moreover, the recent developments and advances of SERS in biomedical application scenarios, such as infectious diseases, cancer diagnostics and therapeutic drug monitoring is given, which enables the readers to identify the sample collection and preparation protocols, SERS substrates and detection strategies that are best-suited for their specific applications in biomedicine.

Determining KRAS4B-Targeting Compound Specificity by Top-Down Mass Spectrometry.

We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform. This method allows us to comprehensively evaluate compound specificity, clearly detect nonspecific binding events, and determine the order and frequency with which they occur. We provide two proof-of-concept examples of our method employing publicly available compounds, along with detailed protocols for sample preparation, top-down MS data acquisition, targeted proteoform MS2 fragmentation, and analysis of the resulting data. Our results demonstrate the concentration dependence of KRAS4B-compound engagement and highlight the ability of top-down MS to directly map compound binding location(s) without disrupting the KRAS4B primary structure. Our hope is that this novel method may help accelerate the identification of new successful targeted inhibitors for KRAS4B and other RAS isoforms.

Chloroform/Methanol Protein Extraction and In-solution Trypsin Digestion Protocol for Bottom-up Proteomics Analysis.

Bottom-up proteomics utilizes sample preparation techniques to enzymatically digest proteins, thereby generating identifiable and quantifiable peptides. Proteomics integrates with other omics methodologies, such as genomics and transcriptomics, to elucidate biomarkers associated with diseases and responses to drug or biologics treatment. The methodologies employed for preparing proteomic samples for mass spectrometry analysis exhibit variability across several factors, including the composition of lysis buffer detergents, homogenization techniques, protein extraction and precipitation methodologies, alkylation strategies, and the selection of digestion enzymes. The general workflow for bottom-up proteomics consists of sample preparation, mass spectrometric data acquisition (LC-MS/MS analysis), and subsequent downstream data analysis including protein quantification and differential expression analysis. Sample preparation poses a persistent challenge due to issues such as low reproducibility and inherent procedure complexities. Herein, we have developed a validated chloroform/methanol sample preparation protocol to obtain reproducible peptide mixtures from both rodent tissue and human cell line samples for bottom-up proteomics analysis. The protocol we established may facilitate the standardization of bottom-up proteomics workflows, thereby enhancing the acquisition of reliable biologically and/or clinically relevant proteomic data. Key features • Tissue/cell pellet sample preparation for bottom-up proteomics. • Chloroform/methanol protein extraction from murine tissue samples. • In-solution trypsin digestion proteomics workflow.

Effect of electron microscopy sample preparation protocol on the preservation of liposomes in cell culture

Effect of electron microscopy sample preparation protocol on the preservation of liposomes in cell culture

Hydrophilic Interaction Liquid Chromatography (HILIC) Enrichment of Glycopeptides Using PolyHYDROXYETHYL A.

Glycosylation of proteins is an important post-translational modification that plays a role in a wide range of biological processes, including immune response, intercellular signaling, inflammation, and host-pathogen interaction. Abnormal protein glycosylation has been correlated with various diseases. However, the study of protein glycosylation remains challenging due to its low abundance, microheterogeneity of glycosylation sites, and low ionization efficiency. During the past decade, several methods for enrichment and for isolation of glycopeptides from biological samples have been developed and successfully employed in glycoproteomics research. In this chapter, we discuss the sample preparation protocol and the strategies for effectively isolating and enriching glycopeptides from biological samples, using PolyHYDROXYETHYL A as a hydrophilic interaction liquid chromatography (HILIC) enrichment technique.