Sample Preparation Strategies Research Articles

Comparison of the Sample Preparation Strategies and Impacts on the Tensile Strength of Gas Shale with Variable Moisture Conditions

Moisture significantly affects the mechanical behavior of gas shale and further determines the hydraulic fracturing performance, as it is more attractive. Nevertheless, batch experiments have usually involved variable methodologies regarding the preparation of moisture-contained shale specimens in the sequence (and/or frequency) of drying and soaking treatments. Accordingly, this work investigates how the preparation methodology influences the test results of moisture-contained shale samples. This study compares three commonly used shale sample preparation strategies for acquiring different moisture contents, that is, “dry-wet”, “dry-wet-dry”, and “wet-dry-wet” strategies, followed by a Brazilian splitting test for the mechanical parameters. The results show that under the same saturation conditions, the longer the soaking time during sample preparation, the higher the degradation degree of shale tensile strength. Meanwhile, prolonged soaking can lead to a more discrete distribution of strength values, and the failure mode may deviate from the Brazilian splitting theory model. Under the combined influence of moisture content and soaking time, the tensile strength of shale decreases approximately linearly with increasing saturation, while the degradation degree increases nonlinearly with increasing saturation, and the degradation rate changes from slow to fast. According to the observation of the microstructure of hydrated shale, prolonged soaking can lead to an increase in the expansion of clay minerals in shale by hydration, resulting in looser and more fragmented internal structure, and further degradation in shale strength. In order to weaken the interference of hydration when studying the effect of moisture content on the tensile strength of shale, the soaking time should be minimized as much as possible during the preparation process.

Trace analysis of 20 antihistamines in milk by ultrahigh performance liquid chromatography coupled with high field quadrupole orbitrap high resolution mass spectrometry followed dispersive micro solid phase extraction

Ultrahigh-performance liquid chromatography coupled with high-field quadrupole Orbitrap high resolution mass spectrometry was used for the separation and determination of 20 antihistamines, and a dispersive micro solid-phase extraction procedure using high-performance absorbing material was developed as a sample preparation strategy for extracting 20 antihistamines from milk. Instrument conditions and key parameters influencing extraction efficiency were investigated to obtain an optimized method. The limit of detection for 20 antihistamines in milk using this method is 0.05 µg/L to 1.0 μg/L. Recoveries are between 80.7% and 108.3%, and the relative standard deviation is less than 15%. It is suitable for confirmatory monitoring and quantitative analysis of 20 antihistamines in milk. The results show that antihistamines in milk may be noteworthy issues for human health and environmental pollution.

Room-temperature fabrication of fluorinated covalent organic polymer @ Attapulgite composite for in-syringe membrane solid-phase extraction and analysis of domoic acid in aquatic products

A novel fluorinated covalent organic polymer @ attapulgite composite (F-COP@ATP) was prepared at room temperature for in-syringe membrane solid-phase extraction (SM-SPE) of domoic acid (DA) in aquatic products. Natural ore ATP has the advantages of low cost, good mechanical strength and abundant hydroxyl group on its surface, and in-situ modified F-COP layer can provide abundant adsorption sites. F-COP@ATP combining the advantages of F-COP and ATP, becomes an ideal adsorbent for DA extracting. Moreover, a high-throughput sample preparation strategy was carried out by using the F-COP@ATP membrane as syringe filter and assembling syringes with a ten-channel injection pump. In addition, the experimental factors were optimized, such as pH of extract, amount of adsorbent, velocity of extraction and desorption, type and volume of desorption solvent. The DA analytical method was established by SM-SPE-HPLC/tandem mass spectrometry. The method had a wide linear range with low limit of detection (0.344 ng/kg) and low limit of quantification (1.14 ng/kg). F-COP@ATP membrane can be reused more than five times. The method realized the analysis of DA in scallop and razor clam samples, which shows its application prospect in practical analysis. This study provided an efficient, low-energy and mild idea for preparing other reusable natural mineral ATP-based composite materials for separation and enrichment, which reduces the experimental cost and is closer to environmental protection and green chemistry to a certain extent.

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.

An innovative sample preparation strategy to visualize the crystalline structure of fiber reinforced thermoplastic composites by polarized optical microscopy

AbstractThis study aims at an innovative sample preparation strategy for fiber reinforced thermoplastic (FRTP) composites analyzed via polarized optical microscopy to visualize the crystalline structure of the matrix resin. Investigating the structure is relevant for understanding the relationship with the mechanical properties. In general, polarized optical microscopy is a powerful technique for visualizing polymer crystalline structures at samples sliced into thin sections below 30 μm. However, analyzing FRTPs remains challenging in absence of suitable methods for the preparation of thin sections owing to the embedded fibers. Herein, the preparation of appropriately thin sections in a novel sequence of steps is shown that may generically be applied to FRTPs. Four different types of FRTPs with major matrix resins (GF/PBT, CF/PA6, CF/PEEK and GF/PP) were successfully processed to thickness of 5 μm or below without fiber loss, resin breakage or delamination between fiber and resin demonstrating the utility of the developed method, which is the significant outcome of the study. To the best of our knowledge, the subsequently obtained polarized optical microscopic images for the first time visualized the crystalline structure of the matrix resin revealing nucleation of crystals from the fiber surface and effects of mold flow on the crystallization behavior.Highlights Thinning of fiber reinforced thermoplastics to thickness of 5 μm or below. Thinning achieved despite fiber volume at industrial applications level. Sample preparation ensures ideal conditions for polarized optical microscopy. Visualization of crystalline structure of the matrix resin for the first time. Potential advancement for spectroscopic and electron microscopic analyses.

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.

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.

Targeted Phosphoproteomics of Human Saliva Extracellular Vesicles via Multiple Reaction Monitoring Cubed (MRM3).

Oral squamous cell carcinoma (OSCC) has become a global health problem due to its increasing incidence and high mortality rate. Early intervention through monitoring of the diagnostic biomarker levels during OSCC treatment is critical. Extracellular vesicles (EVs) are emerging surrogates in intercellular communication through transporting biomolecule cargo and have recently been identified as a potential source of biomarkers such as phosphoproteins for many diseases. Here, we developed a multiple reaction monitoring cubed (MRM3) method coupled with a novel sample preparation strategy, extracellular vesicles to phosphoproteins (EVTOP), to quantify phosphoproteins using a minimal amount of saliva (50 μL) samples from OSCC patients with high specificity and sensitivity. Our results established differential patterns in the phosphopeptide content of healthy, presurgery, and postsurgery OSCC patient groups. Notably, we discovered significantly increased salivary phosphorylated alpha-amylase (AMY) in the postsurgery group compared to the presurgery group. We hereby present the first targeted MS method with extremely high sensitivity for measuring endogenous phosphoproteins in human saliva EVs.

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.

Saliva identification in forensic samples by automated microextraction and intact mass analysis of statherin.

The enzyme α-amylase has long been a commonly targeted protein in serological tests for saliva. While being especially abundant in saliva, α-amylase is detectable in vaginal secretions, sweat, fecal matter, breast milk and other matrices. As a result, assays for α-amylase only provide a presumptive indication of saliva. The availability of mass spectrometry-based tools for the detection of less abundant, but more specific, protein targets (e.g., human statherin) has enabled the development of high confidence assays for human saliva. Sample throughput, however, has traditionally been low due to multi-step workflows for protein extraction, quantitation, enzymatic digestion, solid phase cleanup, and nano-/capillary-based chromatography. Here, we present two novel "direct" single-stage extraction strategies for sample preparation. These feature immunoaffinity purification and reversed-phase solid-phase microextraction in conjunction with intact mass analysis of human statherin for saliva identification. Mass analysis was performed on the Thermo Scientific Q-Exactive™ Orbitrap mass spectrometer with a 10-min analytical run time. Data analysis was performed using Byos® from Protein Metrics. Two sample sets were analyzed with a population of 20 individuals to evaluate detection reliability. A series of casework-type samples were then assayed to evaluate performance in an authentic forensic context. Statherin was confidently identified in 92% and 71% of samples extracted using the immunoaffinity purification and solid phase microextraction approaches, respectively. Overall, immunoaffinity purification outperformed the solid phase microextraction, especially with complex mixtures. In toto, robotic extraction and intact mass spectrometry enable the reliable identification of trace human saliva in a variety of sample types.

Совершенствование схем анализа материалов на основе карбида вольфрама

Using atomic emission spectrometry with inductively coupled plasma and flame version of atomic absorption spectrometry, methods for determining the content of Co, Cr, Ti, V in a wide concentration range from 1·10–3 to n·10 % in tungsten carbide-based materials without matrix separation and using certified solid standard samples with good metrological characteristics were developed. The relative standard deviation is 0.05 – 0.005 when the content of elements is from 1 to 20 % and does not exceed 0.15 when the content of elements is from 0.001 to 0.1 %. Depending on the chemical, phase and disperse composition, as well as the method of obtaining tungsten carbide-based compounds that form different types of materials, which are very different in their ability to dissolve, various sample preparation schemes were used. The analytical parameters for the determination of Co, Cr, Ti, V were found, the operating conditions of the spectrometers were optimized, and calibration methods were selected. The influence of the tungsten matrix element on the analytical signals of analytes and ways of taking it into account have been studied. The possibilities of using spectroscopic methods for the determination of Co, Cr, Ti, V are compared. It is shown that the ICP AES method is dominant in the determination of elements in the concentration range from 1·10–3 to n·10 % in materials based on tungsten carbide. The proposed methods of analytical control will provide research on the creation of new materials based on tungsten carbide, including nanostructured ones, with extremely high physical and mechanical properties, which is especially important in the context of import substitution of materials.

An Updated Guide to the Identification, Quantitation, and Imaging of the Crustacean Neuropeptidome.

Crustaceans serve as a useful, simplified model for studying peptides and neuromodulation, as they contain numerous neuropeptide homologs to mammals and enable electrophysiological studies at the single-cell and neural circuit levels. Crustaceans contain well-defined neural networks, including the stomatogastric ganglion, oesophageal ganglion, commissural ganglia, and several neuropeptide-rich organs such as the brain, pericardial organs, and sinus glands. As existing mass spectrometry (MS) methods are not readily amenable to neuropeptide studies, there is a great need for optimized sample preparation, data acquisition, and data analysis methods. Herein, we present a general workflow and detailed methods for MS-based neuropeptidomic analysis of crustacean tissue samples and circulating fluids. In conjunction with profiling, quantitation can also be performed with isotopic or isobaric labeling. Information regarding the localization patterns and changes of peptides can be studied via mass spectrometry imaging. Combining these sample preparation strategies and MS analytical techniques allows for a multi-faceted approach to obtaining deep knowledge of crustacean peptidergic signaling pathways.

(Invited) Considerations in Sample Environment and Data Processing for Operando Cell Studies by Neutron Depth Profiling

Neutron depth profiling (NDP) is a nominally non-destructive analytical technique used to measure select isotopes (6Li, 14N, 10B, etc...) in a diversity of materials. NDP can be conducted on operating Li-ion cells and be used to provide researchers real-time information about cell formation, conductance, and resilience. The increased use of NDP for electrochemical studies within the past decade has inspired the development of new sample preparation and data reduction strategies. Select topics that have been studied in the U.S. have included issues of neutron attenuation and scattering due to the addition of a protective capping layer over a cell, the possible need for matrix matched standards for complex materials, and the streamlining of the NDP data reduction procedure. Several challenges remain, however, including improving NDP data acquisition and reduction methods for detectable, low-Z isotopes such as 14N and 35Cl and improving upon the NDP data reduction and processing procedures so that they are more accessible to non-instrument specialists. All these topics will be discussed in this presentation.

Untargeted Detection of HIF Stabilizers in Doping Samples: Activity-Based Screening with a Stable In Vitro Bioassay.

Hypoxia-inducible factor (HIF) stabilizers are listed in the World Anti-Doping Agency's prohibited list as they can increase aerobic exercise capacity. The rapid pace of emergence of highly structurally diverse HIF stabilizers could pose a risk to conventional structure-based methods in doping control to detect new investigational drugs. Therefore, we developed a strategy that is capable of detecting the presence of any HIF stabilizer, irrespective of its structure, by detecting biological activity. Previously developed cell-based HIF1/2 assays were optimized to a stable format and evaluated for their screening potential toward HIF stabilizers. Improved pharmacological characterization was established by the stable cell-based formats, and broad specificity was demonstrated by pharmacologically characterizing a diverse set of HIF stabilizers (including enarodustat, IOX2, IOX4, MK-8617, JNJ-42041935). The methodological (in solvent) limit of detection of the optimal HIF1 stable bioassay toward detecting the reference compound roxadustat was 100 nM, increasing to 50-100 ng/mL (corresponding to 617-1233 nM in-well) in matching urine samples, owing to strong matrix effects. In a practical context, a urinary limit of detection of 1.15 μg/mL (95% detection rate) was determined, confirming the matrix-dependent detectability of roxadustat in urine. Pending optimization of a universal sample preparation strategy and/or a methodology to correct for the matrix effects, this untargeted approach may serve as a complementing method in antidoping control, as theoretically, it would be capable of detecting any unknown substance with HIF stabilizing activity.

Synthesis of novel MOF-on-MOF composite as a magnetic sorbent to dispersive micro solid phase extraction of benzodiazepine drugs prior to determination with HPLC-UV

Diazepam and lorazepam are two benzodiazepine drugs widely prescribed as antidepressants and antiepileptics. A straightforward and time-save dispersive micro solid phase extraction method as a sample preparation strategy was developed to extract these drugs in human urine and real water samples prior to their determination by HPLC-UV. A novel sorbent based on growing Co-MOF on Fe-MOF was synthesized and then magnetized using magnetic Fe3O4 nanoparticles and a sol–gel strategy. The sorbent (Fe3O4@SiO2@Co-MOF@Fe-MOF composite) showed the highest extraction efficiency toward diazepam and lorazepam compared to Fe-MOF and Co-MOF@Fe-MOF as sorbents. The effects of various factors on diazepam and lorazepam determination were studied by experimental design, in which a Plackett-Burman design and Box-Behnken design were used for screening the factors and optimizing the significant factors, respectively. Under optimum conditions, the method linearity for the diazepam and lorazepam determination was 0.3–450 and 0.2–400 ng mL−1 with an R2 of 0.9926 and 0.9943, respectively. Proper detection limits of 0.09 and 0.06 ng mL−1 were obtained for the diazepam and lorazepam determination. Also, intra-day and inter-day RSDs (n = 3) were in the range of 3.42–3.75 % and 4.76–5.39 % for the diazepam and lorazepam determination, respectively. The method exhibits a high preconcentration factor of 654.3 and 719.1 for the diazepam and lorazepam determination. Besides, the recoveries and RSDs were evaluated by analysis of tap, river, and well water, and two human urine samples using the method. RSDs were in the range of 3.09–6.80 %, with recoveries between 88.2 and 97.5 % for analysis of spiked real water and urine samples.

Quality control of wood treated with citric acid and sorbitol using a handheld Raman spectrometer

Wood modification using polyesterification of sorbitol and citric acid is a novel environmentally friendly strategy for wood protection improving its dimensional stability and acts against fungal deterioration. Inelastic Raman scattering is sensitive to the molecules of high polarizability and both lignocellulose and aliphatic esters formed during the treatment are polar. Therefore, in the present study, the quality control of the treatment using a handheld Raman spectrometer equipped with 830 nm laser is suggested as a rapid and reliable approach. Raman spectra from six wood modification levels (resulting in different weight percent gain, WPG) of three different wood species (Silver birch, Scots pine and Norway spruce) as well as three sample preparation strategies (intact, sanded and milled wood samples) were collected, and further analyzed using a chemometric method. Best performing models based on Powered Partial Least Squares Regression predicted the WPG level at R2 = 0.85, 0.95 and 0.98 for birch, pine and spruce, respectively. In addition, a clear separation between hard and soft wood species was also captured. Especially for softwood species, the sample preparation method affected the model accuracy, revealing the best performance in milled material. It is concluded that by using handheld Raman spectrometer it is possible to perform accurate quality control of wood modified by polyesterification of citric acid and sorbitol.

RUPE-phospho: Rapid Ultrasound-Assisted Peptide-Identification-Enhanced Phosphoproteomics Workflow for Microscale Samples.

Global phosphoproteome profiling can provide insights into cellular signaling and disease pathogenesis. To achieve comprehensive phosphoproteomic analyses with minute quantities of material, we developed a rapid and sensitive phosphoproteomics sample preparation strategy based on ultrasound. We found that ultrasonication-assisted digestion can significantly improve peptide identification by 20% due to the generation of longer peptides that can be detected by mass spectrometry. By integrating this rapid ultrasound-assisted peptide-identification-enhanced proteomic method (RUPE) with streamlined phosphopeptide enrichment steps, we established RUPE-phospho, a fast and efficient strategy to characterize protein phosphorylation in mass-limited samples. This approach dramatically reduces the sample loss and processing time: 24 samples can be processed in 3 h; 5325 phosphosites, 4549 phosphopeptides, and 1888 phosphoproteins were quantified from 5 μg of human embryonic kidney (HEK) 293T cell lysate. In addition, 9219 phosphosites were quantified from 1-2 mg of OCT-embedded mouse brain with 120 min streamlined RUPE-phospho workflow. RUPE-phospho facilitates phosphoproteome profiling for microscale samples and will provide a powerful tool for proteomics-driven precision medicine research.

Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay

In the ongoing global fight against coronavirus disease 2019 (COVID-19), the sample preparation process for real-time reverse transcription polymerase chain reaction (rRT-PCR) faces challenges due to time-consuming steps, labor-intensive procedures, contamination risks, resource demands, and environmental implications. However, optimized strategies for sample preparation have been poorly investigated, and the combination of RNase inhibitors and Proteinase K has been rarely considered. Hence, we investigated combinations of several extraction-free protocols incorporating heat treatment, sample dilution, and Proteinase K and RNase inhibitors, and validated the effectiveness using 120 SARS-CoV-2 positive and 62 negative clinical samples. Combining sample dilution and heat treatment with Proteinase K and RNase inhibitors addition exhibited the highest sensitivity (84.26%) with a mean increase in cycle threshold (Ct) value of + 3.8. Meanwhile, combined sample dilution and heat treatment exhibited a sensitivity of 79.63%, accounting for a 38% increase compared to heat treatment alone. Our findings highlight that the incorporation of Proteinase K and RNase inhibitors with sample dilution and heat treatment contributed only marginally to the improvement without yielding statistically significant differences. Sample dilution significantly impacts SARS-CoV-2 detection, and sample conditions play a crucial role in the efficiency of extraction-free methods. Our findings may provide insights for streamlining diagnostic testing, enhancing its accessibility, cost-effectiveness, and sustainability.

A Guide to MALDI Imaging Mass Spectrometry for Tissues.

Imaging mass spectrometry is a well-established technology that can easily and succinctly communicate the spatial localization of molecules within samples. This review communicates the recent advances in the field, with a specific focus on matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) applied on tissues. The general sample preparation strategies for different analyte classes are explored, including special considerations for sample types (fresh frozen or formalin-fixed,) strategies for various analytes (lipids, metabolites, proteins, peptides, and glycans) and how multimodal imaging strategies can leverage the strengths of each approach is mentioned. This work explores appropriate experimental design approaches and standardization of processes needed for successful studies, as well as the various data analysis platforms available to analyze data and their strengths. The review concludes with applications of imaging mass spectrometry in various fields, with a focus on medical research, and some examples from plant biology and microbe metabolism are mentioned, to illustrate the breadth and depth of MALDI IMS.

An ionic liquid-assisted sample preparation method for sensitive integral-membrane proteome analysis

Many ion channels and receptor proteins are potential targets for new drugs. However, standard methods for profiling these integral membrane proteins (IMPs) have not been fully established, especially when applied to rare and quantity-limited biological samples. We previously demonstrated that a mixture containing 1-butyl-3-methylimidazolium cyanate, an ionic liquid (IL), and NaOH (termed i-soln) is an excellent solubilizer for insoluble aggregates. In this study, we present a combined i-soln-assisted proteomic sample preparation platform (termed pTRUST), which is compatible with starting materials in the sub-microgram range, using our previously reported i-soln-based sample preparation strategy (iBOPs) and an in-StageTip technique. This novel and straightforward approach allows for the rapid solubilization and processing of a variety of IMPs from human samples to support highly sensitive mass spectrometry analysis. We also demonstrated that the performance of this technology surpasses that of conventional methods such as filter-aided sample preparation methods, FASP and i-FASP. The convenience and availability of pTRUST technology using the IL system have great potential for proteomic identification and characterization of novel drug targets and disease biology in research and clinical settings.