Sample Preparation Strategies Research Articles

Study of sample preparation for determination of endocannabinoids and analogous compounds in human serum by LC–MS/MS in MRM mode

Endocannabinoids are lipids with a key role in physiological processes such as the immune response or the metabolism. This involvement explains their association to pathologies such as cancer, obesity or multiple sclerosis. The determination of endocannabinoids constitutes a challenge for clinical laboratories due to the variety of biological matrices and the wide range of concentrations at which they can be found. This research deals with the comparison of three sample preparation strategies (viz., on-line SPE, off-line SPE for interferents removal, and protein precipitation) for subsequent LC–MS/MS analysis of 14 endocannabinoids and analogous compounds in serum. As a result, the on-line coupling between SPE and LC–MS/MS is proposed as the best approach for this determination. The proposed method allows full automation of the overall process, shortening of the analysis time, and avoidance of errors associated with sample preparation steps. The improvement in sensitivity and selectivity thus achieved allows obtaining quantification limits at the pg mL−1 level, which makes possible the application of the method for clinical studies.

Assessment of Sample Preparation Bias in Mass Spectrometry-Based Proteomics.

For mass spectrometry-based proteomics, the selected sample preparation strategy is a key determinant for information that will be obtained. However, the corresponding selection is often not based on a fit-for-purpose evaluation. Here we report a comparison of in-gel (IGD), in-solution (ISD), on-filter (OFD), and on-pellet digestion (OPD) workflows on the basis of targeted (QconCAT-multiple reaction monitoring (MRM) method for mitochondrial proteins) and discovery proteomics (data-dependent acquisition, DDA) analyses using three different human head and neck tissues (i.e., nasal polyps, parotid gland, and palatine tonsils). Our study reveals differences between the sample preparation methods, for example, with respect to protein and peptide losses, quantification variability, protocol-induced methionine oxidation, and asparagine/glutamine deamidation as well as identification of cysteine-containing peptides. However, none of the methods performed best for all types of tissues, which argues against the existence of a universal sample preparation method for proteome analysis.

Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis

Background:Current syphilis diagnostic strategies are lacking a sensitive manner of directly detectingTreponema pallidumantigens. A diagnostic test that could directly detectT. pallidumantigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up.Methods:In this study, 11 candidateT. pallidumbiomarker proteins were chosen according to their physiochemical characteristics,T. pallidumspecificity and predicted abundance. Thirty isotopically labelled proteotypic surrogate peptides (hPTPs) were synthesized and incorporated into a scheduled multiple reaction monitoring assay. Protein extracts from undepleted/unenriched plasma (N = 18) and urine (N = 4) samples from 18 individuals with syphilis in various clinical stages were tryptically digested, spiked with the hPTP mixture and analysed with a triple quadruple mass spectrometer.Results:No endogenous PTPs corresponding to the eleven candidate biomarkers were detected in any samples analysed. To estimate the Limit of Detection (LOD) of a comparably sensitive mass spectrometer (LTQ-Orbitrap), two dilution series of rabbit cultured purifiedT. pallidumwere prepared in PBS. Polyclonal anti-T. pallidumantibodies coupled to magnetic Dynabeads were used to enrich one sample series; no LOD improvement was found compared to the unenriched series. The estimated LOD of MS instruments is 300T. pallidum/ml in PBS.Conclusions:Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration ofT. pallidumproteins. Alternative sample preparation strategies may improve the detectability ofT. pallidumproteins in biofluids.

Lipidomic Analysis.

The state-of-art in the lipidomic analysis is summarized here to provide the overview of available sample preparation strategies, mass spectrometry (MS)-based methods for the qualitative analysis of lipids, and the quantitative MS approaches for high-throughput clinical workflows. Major challenges in terms of widely accepted best practices for lipidomic analysis, nomenclature, and standards for data reporting are discussed as well.

Determination of trace metals in spirits by total reflection X-ray fluorescence spectrometry

Eight spirituous samples were analyzed for trace metal content with Horizon Total Reflection X-Ray Fluorescence (TXRF) Spectrometer. The expected single metal amount is at the ng/g level in a mixed aqueous/organic matrix, thus requiring a sample preparation method capable of achieving suitable limits of detection. On-site enrichment and Atmospheric Pressure-Vapor Phase Decomposition allowed to detect Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Sr and Pb with detection limits ranging from 0.1ng/g to 4.6ng/g. These results highlight how the synergy between instrument and sample preparation strategy may foster the use of TXRF as a fast and reliable technique for the determination of trace elements in spirituous samples, either for quality control or risk assessment purposes.

Optimized method for determination of 16 FDA polycyclic aromatic hydrocarbons (PAHs) in mainstream cigarette smoke by gas chromatography\u2013mass spectrometry

A gas chromatography–mass spectrometry (GC–MS) method was validated for the determination of 16 polycyclic aromatic hydrocarbons (PAHs) from the FDA list of 93 harmful or potentially harmful constituents of mainstream cigarette smoke (MCS). Target analytes were extracted from total particulate matter using accelerated solvent extraction with a toluene/ethanol solvent mixture. Matrix artefacts were removed by two-step solid-phase extraction process. Three different GC–MS systems [GC–MS (single quadrupole), GC–MS/MS (triple quadrupole) and GC–HRMS (high resolution, magnetic sector)] using the same separation conditions were compared for the analysis of MCS of 3R4F Kentucky reference cigarettes generated under ISO and intense smoking regimes. The high mass resolution (m/∆m ≥ 10,000) and associated selectivity of detection by GC–HRMS provided the highest quality data for the target PAHs in MCS. Owing to the HR data acquisition mode enabling measurement of accurate mass, limits of quantification for PAHs were 5 to 15-fold lower for GC–HRMS than for GC–MS/MS and GC–MS. The presented study illustrates that the optimised sample preparation strategy followed by GC–HRMS analysis provides a fit-for-purpose and robust analytical approach allowing measurement of PAHs at (ultra)low concentrations in MCS. Furthermore, the study illustrates the importance and benefits of robust sample preparation and clean-up to compensate for limited selectivity when low-resolution MS is used.

A Streamlined Sample Preparation Method for Mass Spectrometric Analysis.

Mass spectrometry-based proteomic technology experienced remarkable advancement in the past decades. However, their application was still hampered by the complexity of sample preparation. Conventional strategies for sample preparation incorporate multiple time-consuming steps, including cell lysis, protein extraction, protease cleavage, and desalting. Thus, we explored a simplified method (the cell-absorb method) during which living cells were absorbed into vacuum-dried polyacrylamide gel and directly digested in gel into peptides for subsequent LC-MS/MS analysis. As a consequence, both of the steps for cell lysis and protein extraction involved in traditional protocol were skipped. In addition to the decrease in time, more proteins were identified. Indeed, 3022 proteins were identified by the cell-absorb method. Meanwhile, only 2642 and 2420 proteins were identified by the classical SDS-PAGE based method and the reported gel absorption-based method, respectively. The cell-absorb method exhibited apparent advantage in terms of the depth of proteome coverage. Furthermore, the number of proteins identified show excellent reproducibility with a CV (coefficient of variation) of 0.03 among three replicates using the cell-absorb method. These advantages suggest that cell-absorb method is a promising choice for mapping the whole proteome of cells. © 2018 by John Wiley & Sons, Inc.

Sensitive determination of brassinosteroids by solid phase boronate affinity labeling coupled with liquid chromatography-tandem mass spectrometry

Brassinosteroids (BRs) are regarded as the sixth plant hormone that is widely distributed in the plant kingdom. Sensitive quantification of BRs will be greatly benefit to illuminate the detail mechanisms about how BRs play crucial role in plant developmental processes such as cell division, cell expansion, cytodifferentiation, seed germination, vegetative growth and resisting biological or abiotic stress. In the current study, we developed a method for rapid and sensitive determination of endogenous BRs in plant tissues by combining LC–MS and a novel sample preparation strategy, in which the plant tissue extract was supplied to solid phase boronate affinity labeling and extraction, followed by desorption and salt-induced phase transition extraction for further purification. Under the optimized conditions, good linearity was obtained for 6 BR with correlation coefficients (r) ranging from 0.9988 to 0.9999. The limits of detection (LODs, S/N=3) ranged from 1.4 to 2.8pgmL−1. The recoveries were between 93.4% and 116.2% with the relative standard deviations (RSDs) ranging from 2.8% to 15.8%. Finally, the developed method was successfully applied to the analysis of 6 endogenous BR in various plant tissues including 20mg FW Oryza sativa shoot, 10mg FW Oryza sativa root, 20mgFW Arabidopsis thaliana shoot, 4 Arabidopsis thaliana flowers (2.8mg) and one Brassica napus stamen (3.0mg) with concentration ranging from 0.26 to 157.28ngg−1FW.

RNA Characterization by Solid-State NMR Spectroscopy.

The structures of RNAs, which play critical roles in various biological processes, provide important clues and insights into the biological functions of these molecules. However, RNA structure determination remains a challenging topic. In recent years, magic-angle-spinning solid-state NMR (MAS SSNMR) has emerged as an alternative technique for structural and dynamic characterization of RNA. MAS SSNMR has been successfully applied to provide atomic-level structural information about several RNA molecules and RNA-protein complexes. In this Minireview, we give an overview of recent progress in the field of MAS SSNMR based RNA structural characterization, and introduce sample preparation strategies and SSNMR spectroscopic techniques that have been incorporated to identify RNA structural elements. We also highlight a few impressive examples of RNAs that have been investigated extensively by SSNMR. Finally, we briefly discuss future technical trends in the use of MAS SSNMR to facilitate RNA structure determination.

Detection of nanoplastics in food by asymmetric flow field-flow fractionation coupled to multi-angle light scattering: possibilities, challenges and analytical limitations

We tested the suitability of asymmetric flow field-flow fractionation (AF4) coupled to multi-angle light scattering (MALS) for detection of nanoplastics in fish. A homogenized fish sample was spiked with 100nm polystyrene nanoparticles (PSNPs) (1.3mg/g fish). Two sample preparation strategies were tested: acid digestion and enzymatic digestion with proteinase K. Both procedures were found suitable for degradation of the organic matrix. However, acid digestion resulted in large PSNPs aggregates/agglomerates (> 1μm). The presence of large particulates was not observed after enzymatic digestion, and consequently it was chosen as a sample preparation method. The results demonstrated that it was possible to use AF4 for separating the PSNPs from the digested fish and to determine their size by MALS. The PSNPs could be easily detected by following their light scattering (LS) signal with a limit of detection of 52μg/g fish. The AF4-MALS method could also be exploited for another type of nanoplastics in solution, namely polyethylene (PE). However, it was not possible to detect the PE particles in fish, due to the presence of an elevated LS background. Our results demonstrate that an analytical method developed for a certain type of nanoplastics may not be directly applicable to other types of nanoplastics and may require further adjustment. This work describes for the first time the detection of nanoplastics in a food matrix by AF4-MALS. Despite the current limitations, this is a promising methodology for detecting nanoplastics in food and in experimental studies (e.g., toxicity tests, uptake studies). Graphical abstract Basic concept for the detection of nanoplastics in fish by asymmetric flow field-flow fractionation coupled to multi-angle light scattering.

Simultaneous accurate quantification of HO-1, CD39, and CD73 in human calcified aortic valves using multiple enzyme digestion – filter aided sample pretreatment (MED-FASP) method and targeted proteomics

Several proteins such as membrane-associated ectonucleotidases: ecto-5’-nucleotidase (E5NT/CD73) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), and intracellular heme oxygenase-1 (HO-1) may contribute to protection from inflammation-related diseases such as calcific aortic valve stenosis (CAS). Accurate quantification of these proteins could contribute to better understanding of the disease mechanisms and identification of biomarkers.This report presents development and validation of quantification method for E5NT/CD73, ENTPD1/CD39 and HO-1. The multiplexed targeted proteomic assay involved antibody-free, multiple-enzyme digestion, filter-assisted sample preparation (MED-FASP) strategy and a nanoflow liquid chromatography/mass spectrometry under multiple reaction monitoring mode (LC-MRM/MS). The method developed presented high sensitivity (LLOQ of 5 pg/mL for each of the analytes) and accuracy that ranged from 92.0% to 107.0%, and was successfully applied for the absolute quantification of HO-1, CD39 and CD73 proteins in homogenates of human calcified and non-calcified valves. The absolute CD39 and CD73 concentrations were lower in calcified aortic valves (as compared to non-stenotic ones) and were found to be: 1.16 ± 0.39 vs. 3.15 ± 0.37 pmol/mg protein and 1.94 ± 0.21 vs. 2.39 ± 0.39 pmol/mg protein, respectively, while the quantity of HO-1 was elevated in calcified valves (10.72 ± 1.18 vs. 4.28 ± 0.42 amol/mg protein). These results were consistent but more reproducible as compared to immunoassays.In conclusion, multiplexed quantification of HO-1, CD39 and CD73 proteins by LC-MRM/MS works well in challenging human tissues such as aortic valves. This analysis confirmed the relevance of these proteins in pathogenesis of CAS and could be extended to other biomedical investigations.

Development of an enrichment method for endogenous phosphopeptide characterization in human serum.

The work describes the development of an enrichment method for the analysis of endogenous phosphopeptides in serum. Endogenous peptides can play significant biological roles, and some of them could be exploited as future biomarkers. In this context, blood is one of the most useful biofluids for screening, but a systematic investigation of the endogenous peptides, especially phosphorylated ones, is still lacking, mainly due to the lack of suitable analytical methods. Thus, in this paper, different phosphopeptide enrichment strategies were pursued, based either on metal oxide affinity chromatography (MOAC, in the form of commercial TiO2 spin columns or magnetic graphitized carbon black-TiO2 composite), or on immobilized metal ion affinity chromatography (IMAC, in the form of Ti4+-IMAC magnetic material or commercial Fe3+-IMAC spin columns). While MOAC strategies proved completely unsuccessful, probably due to interfering phospholipids displacing phosphopeptides, the IMAC materials performed very well. Different sample preparation strategies were tested, comprising direct dilution with the loading buffer, organic solvent precipitation, and lipid removal from the matrix, as well as the addition of phosphatase inhibitors during sample handling for maximized endogenous phosphopeptide enrichment. All data were acquired by a shotgun peptidomics approach, in which peptide samples were separated by reversed-phase nanoHPLC hyphenated with high-resolution tandem mass spectrometry. The devised method allowed the identification of 176 endogenous phosphopeptides in fresh serum added with inhibitors by the direct dilution protocol and the Ti4+-IMAC magnetic material enrichment, but good results could also be obtained from the commercial Fe3+-IMAC spin column adapted to the batch enrichment protocol.

LC-MS/MS Analysis of Triglycerides in Blood-Derived Samples.

The increasing interest in the analysis of triglyceride (TG) species and the individual fatty acid (FA) composition requires expeditious and reliable quantification strategies. The utilization of flow injection analysis (FIA) coupled to quadrupole tandem mass spectrometry (MS/MS) for the simultaneous quantitation of TG and identification of FA composition facilitates the multiplexed verification of various biomarkers from small sample quantities. Enzymatic methods based on saponification and glycerol analysis are not suited for the determination of the FA distribution in TGs. This protocol proposes a procedure for the establishment of a relative quantitation method for middle- to high-abundance plasma TGs and the corresponding FA composition. Essential topics as FIA-MS/MS method development as well as sample preparation and validation strategies are described in detail.

Sample preparation strategies for petroleum coke digestion and further cerium and lanthanum determination by DSN-ICP-OES

Digestion of petroleum coke is proposed for the first time by MW-AD and MIC for La and Ce determination by DSN-ICP-OES.

Identification, Quantitation, and Imaging of the Crustacean Peptidome.

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. In particular, crustaceans contain well-defined neural networks, including the stomatogastric ganglion, esophageal ganglion, commissural ganglia, and several neuropeptide-rich organs, such as the brain, pericardial organs, and sinus glands. Due to the lack of a genomic database for crustacean peptides, an important step of crustacean peptidomics involves the discovery and identification of novel peptides and the construction of a database, more recently with the aid of mass spectrometry (MS). Herein, we present a general workflow and detailed methods for MS-based peptidomic 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 multifaceted approach to obtaining deep knowledge of crustacean peptidergic signaling pathways.

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) coupled with direct computational fitting of the observed concentration profiles (sedimentating boundary) have been developed and widely used for the characterization of macromolecules and nanoparticles in solution. In particular, size distribution analysis by SV-AUC has become a reliable and essential approach for the characterization of biopharmaceuticals including therapeutic antibodies. In this review, we describe the importance and advantages of SV-AUC for studying biopharmaceuticals, with an emphasis on strategies for sample preparation, data acquisition, and data analysis. Recent discoveries enabled by AUC with a fluorescence detection system and potential future applications are also discussed.

AFM-Raman colocalization setup: Advanced characterization technique for polymers

ABSTRACTThe optimization of manufacturing processes and the investigation of polymer materials during their life cycle imply an extended knowledge of the physical and chemical properties of the material. To obtain colocalized chemical, morphological, and thermomechanical information on the same sample area, we have developed an innovative colocalized atomic force microscopy (AFM)-Raman setup and a sample preparation strategy using cryoultramicrotomy. The developed technique allows perfect cross-sectioning of thin membranes (10–300 µm) without embedding avoiding chemical contamination. The opened surface is characteristic of the material’s bulk properties and allows spectroscopic and near-field measurements without sample artifacts. Additionally, thin sections can be collected for complementary transmission electron microscopy.

Influence of different sample preparation strategies on the proteomic identification of stress biomarkers in porcine saliva

BackgroundThe influence of two different sample treatments comprising the enrichment of glycoproteins by boronic acid and dynamic range compression by hexapeptide libraries, on the detection of stress markers in saliva of pigs was evaluated in this study. For this purpose, saliva samples collected before and after the application of an acute stress model consisting of nasal restraining in pigs were processed without any treatment and with the two different treatments mentioned above. Protein separation by two-dimensional gel electrophoresis (2-DE) followed by identification of proteins using MALDI-TOF/TOF mass spectrometry (MS) was used as proteomic technique.ResultsThe application of each of the two different sample treatment protocols allowed the identification of unique proteins that could be potential salivary acute stress markers in pigs: lipocalin 1, protein S100-A8 and immunoglobulin M by enrichment of glycoproteins; protein S100-A9, double headed protease inhibitor submandibular gland, and haemoglobin by dynamic range compression; and protein S100-A12 by both protocols. Salivary lipocalin, prolactin inducible protein, light chain of immunoglobulins, adenosine deaminase and carbonic anhydrase VI were identified as potential markers in untreated saliva as well as one of the other treatments.ConclusionThe use of different procedures allowed the detection of different potential stress markers. Although from a practical point of view, the use of saliva without further treatment as well as the enrichment of glycoproteins are less expensive and easy to do procedures.

Filter Plate–Based Screening of MIP SPE Materials for Capture of the Biomarker Pro-Gastrin-Releasing Peptide

Affinity-based solid-phase extraction (SPE) is an attractive low-cost sample preparation strategy for biomarker analysis. Molecularly imprinted polymers (MIPs) as affinity sorbents offer unique opportunities for affinity SPE, due to their low manufacturing cost and high robustness. A limitation is the prediction of their affinity; therefore, screening of analyte recovery and specificity within a large range of SPE conditions is important in order to ensure high-sensitivity detection and assay reproducibility. Here, a µ-SPE method for screening of the MIP-SPE materials using a commercial 384-well filter plate is presented. The method allows for rapid and automated screening using 10–30 µL of packed SPE sorbent per well and sample volumes in the range of 10–70 µL. This enables screening of many different SPE sorbents while simultaneously identifying optimal SPE conditions. In addition, the 384-well format also facilitates detection with a multitude of analytical platforms. Performance of the µ-MIP-SPE method was investigated using a series of MIPs designed to capture pro-gastrin-releasing peptide (ProGRP). Fractions coming from sample load, cartridge wash, and elution were collected and analyzed using mass spectrometry (MS). The top-performing MIPs were identified, together with proper SPE conditions.

Proteomics and the human microbiome: where we are today and where we would like to be.

What are all these hundreds of different bacterial species doing in and on us? What interactions occur between the host and the microbes, and between the microbes themselves? By studying proteins, metaproteomics tries to find preliminary answers to these questions. There is daunting complexity around this; in fact, many of these proteins have never been studied before. This article is an introduction to the field of metaproteomics in the context of the human microbiome. It summarizes where we are and what we have learnt so far. The focus will be on faecal proteomics as most metaproteomics research has been conducted on that sample type. Metaproteomics has made major advances in the past decade, but new sample preparation strategies, improved mass spectrometric analysis and, most importantly, data analysis and interpretation have the potential to pave the way for large-cohort metaproteomics.