Direct Analysis Of Biological Samples Research Articles

Applications of MALDI-MS/MS-Based Proteomics in Biomedical Research.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is one of the most widely used techniques in proteomics to achieve structural identification and characterization of proteins and peptides, including their variety of proteoforms due to post-translational modifications (PTMs) or protein–protein interactions (PPIs). MALDI-MS and MALDI tandem mass spectrometry (MS/MS) have been developed as analytical techniques to study small and large molecules, offering picomole to femtomole sensitivity and enabling the direct analysis of biological samples, such as biofluids, solid tissues, tissue/cell homogenates, and cell culture lysates, with a minimized procedure of sample preparation. In the last decades, structural identification of peptides and proteins achieved by MALDI-MS/MS helped researchers and clinicians to decipher molecular function, biological process, cellular component, and related pathways of the gene products as well as their involvement in pathogenesis of diseases. In this review, we highlight the applications of MALDI ionization source and tandem approaches for MS for analyzing biomedical relevant peptides and proteins. Furthermore, one of the most relevant applications of MALDI-MS/MS is to provide “molecular pictures”, which offer in situ information about molecular weight proteins without labeling of potential targets. Histology-directed MALDI-mass spectrometry imaging (MSI) uses MALDI-ToF/ToF or other MALDI tandem mass spectrometers for accurate sequence analysis of peptide biomarkers and biological active compounds directly in tissues, to assure complementary and essential spatial data compared with those obtained by LC-ESI-MS/MS technique.

In-Syringe Electrokinetic Protein Removal from Biological Samples prior to Electrospray Ionization Mass Spectrometry.

Here, an electrokinetic extraction (EkE) syringe is presented allowing for on-line electrokinetic removal of serum proteins before ESI-MS. The proposed concept is demonstrated by the determination of pharmaceuticals from human serum within minutes, with sample preparation limited to a 5× dilution of the sample in the background electrolyte (BGE) and application of voltage, both of which can be performed in-syringe. Signal enhancements of 3.6-32 fold relative to direct infusion of diluted serum and up to 10.8 fold enhancement, were obtained for basic and acidic pharmaceuticals, respectively. Linear correlations for the basic drugs by EkE-ESI-MS/MS were achieved, covering the necessary clinical range with LOQs of 5.3, 7.8, 6.1, and 17.8 ng mL-1 for clomipramine, chlorphenamine, pindolol, and atenolol, respectively. For the acidic drugs, the EkE-ESI-MS LOQs were 3.1 μg mL-1 and 2.9 μg mL-1 for naproxen and paracetamol, respectively. The EkE-ESI-MS and EkE-ESI-MS/MS methods showed good accuracy (%found of 81 % to 120 %), precision (≤20 %), and linearity (r>0.997) for all the studied drugs in spiked serum samples.

Detection of Nerve Agent Metabolites in Plasma and Urine by 31P NMR Spectroscopy

A simple robust 31P nuclear systems based NMR method is presented for the detection of alkyl methylphosphonic acids; metabolites of three organophosphorus nerve agents (GB or Sarin, GD or Soman, and GF or Cyclosarin) in the biological spiked samples of plasma and urine. The method gives linear response (correlation coefficients ~0.89) over the concentration range 10 μg/ml to 300 μg/ml in urine and plasma considering phosphorus nucleus. The quality control data demonstrated accurate identification of biomarkers of nerve agents at low concentration. NMR based method provides direct analysis of Biological samples without any sample pretreatment or sample preparation and sample tempering.

High ohmic resistor hyphenated gel loading tip nano-electrospray ionization source for mini mass spectrometer

The deployment of mini mass spectrometers on the field strongly demands efficient ionization sources that are easy-to-operate. Nano-electrospray (nESI) ion source has been widely used in the field of chemistry, biology, medicine, pharmaceutical industry, clinical assessment and forensic science. In this study, a high ohmic resistor hyphenated gel loading tip nESI source was coupled with our home developed mini mass spectrometer. This ionization source has the advantages of simple-in-design, disposable and low-in-cost, therefore it could be frequently used for analysis of aqueous samples without leading to cross contamination. Performances of the gel loading tip nESI emitter were similar to pulled glass capillary, and highly compatible for the analysis of biomolecule in aqueous solution. Different peptide and small molecules have been confirmed with a continuous atmospheric pressure-interfaced (CAPI) mini mass spectrometer. The corona discharge, which was usually observed at nESI emitter tip under high aqueous solvent conditions, resulting in low ion intensity, has been successfully quenched using a 10 GΩ resistor in both a pulled glass capillary and a gel loading tip as nESI emitter in this study. Compared with conventional ESI, the metal wire assisted gel loading tip facilitated loading and direct analysis of biological samples without sample pretreatment.

Validated Microemulsion Liquid Chromatography-Fluorescence Method for the Quantification of Duloxetine and its Two Main Metabolites in Plasma: Application to Clinical Pharmacokinetic Studies

Background: Duloxetine (DL) is a selective serotonin and norepinephrine reuptake inhibitor. The drug is used in the treatment of major depression, anxiety, pain related to diabetic peripheral neuropathy and stress urinary incontinence. </P><P> Objective: This study described, for the first time, the development and validation of a highly selective and sensitive microemulsion liquid chromatography-fluorescence (MELC-FL) method with low environmental pollution and without extraction steps for the simultaneous quantification of DL, and its two main metabolites; 5-hydroxy-6-methoxy duloxetine (5-HDL) and 4-hydroxy duloxetine glucuronide (4- HDLG) in plasma. Methods: The studied analytes and methyl paraben (an internal standard) were detected using excitation and emission wavelengths of 280 and 340 nm, respectively. The analysis was performed on Water Symmetry C18 analytical column (100 Å, 150 mm x 3.9 mm, 5 µm) by directly injecting the plasma after appropriate dilution with microemulsion mobile phase. Total analytical run time was 4 min. Results: The MELC-FL method was statistically validated according to the FDA guidelines for bioanalytical methods for linearity, accuracy, precision, specificity, robustness, and stability. Linear calibration plots were achieved in the ranges of 25-1200 ng/mL for DL and 50-1500 ng/mL for 5-HDL and 4- HDLG (r2 ≥ 0.997) in rat plasma. The intra- and inter- assay precisions and accuracy were acceptable. The overall recoveries of DL and its two main metabolites from rat plasma were between 97.12% and 103.12% with an RSD value between 0.34% and 4.57%. Conclusion: The present study supports the possible use of the microemulsion mobile phase in LC as a “greener ” mobile phase. The developed method offered an advantage in the form of direct analysis of biological samples after appropriate dilution with eco-friendly microemulsion mobile phase, which decreased the possibility of sample loss during analysis. The developed assay was successfully applied in a pharmacokinetic study and it established the applicability of the method for the determination of concentration-time profiles of DL and its two main metabolites in rat plasma after systemic administration.

Solvent gradient electrospray for laser ablation electrospray ionization mass spectrometry.

Most electrospray based ambient ionization techniques, e.g., laser ablation electrospray ionization (LAESI), utilize a fixed spray solution composition. Complex samples often contain compounds of different polarity that exhibit a wide range of solubilities in the electrospray solvent. Thus, the fixed spray solution composition limits the molecular coverage of these approaches. Two-barrel theta glass capillaries have been used for the rapid mixing of two solutions for manipulating fast reactions including protein folding, unfolding, and charge state distributions. Here, we present a new variant of LAESI mass spectrometry (MS) by scanning the high voltages applied to the two barrels of a theta glass capillary containing two different solvents. In the resulting gradient LAESI (g-LAESI), the composition of the spray solution is ramped between the two solvents in the barrels to facilitate the detection of compounds of diverse polarity and solubility. Dynamic ranges and limits of detection achieved for g-LAESI-MS were comparable to conventional LAESI-MS. We have demonstrated simultaneous detection of different types of chemical standards, and polar and less polar compounds from Escherichia coli cell pellets using g-LAESI-MS. Varying the spray solution composition in a gradient electrospray can benefit from the enhanced solubilities of different analytes in polar and less polar solvents, ultimately improving the molecular coverage in the direct analysis of biological samples.

Paper-capillary spray for direct mass spectrometry analysis of biofluid samples.

Paper spray has been developed as an ambient ionization method for direct analysis of biological samples using mass spectrometry. While distinct advantages of paper spray have been demonstrated, especially for quantitative analysis and design of disposable sample cartridges, the need for improvement has also been recognized, especially for the use with miniature mass spectrometers. In this study, we made an improvement to the sampling and ionization by adding a capillary emitter to the paper substrate to produce a paper-capillary spray, which has been shown to have significant, positive impact on the sensitivity and reproducibility for direct mass spectrometry analysis. The paper-capillary devices were fabricated and the effects of the geometry, the treatment of the capillary emitters, as well as the sample disposition methods were characterized. The method's analytical performance was also characterized for analysis of therapeutic drugs in blood samples. Quantitation of cotinine in blood using a commercial triple quadrupole and sitagliptin (Januvia®) in blood using a desktop Mini 12 ion trap mass spectrometer was also demonstrated.

Restricted accessed nanoparticles for direct magnetic solid phase extraction of trace metal ions from human fluids followed by inductively coupled plasma mass spectrometry detection.

Herein, restricted accessed magnetic nanoparticles were synthesized by self-assembly of a non-ionic surfactant (Tween-20) onto the 4-(2-pyridylazo)resorcinol (PAR) functionalized magnetic nanoparticles (MNPs). A series of analytical techniques were employed for the characterization of the as-prepared restricted accessed Fe3O4@SiO2@PAR, and it was found that the as-prepared restricted accessed Fe3O4@SiO2@PAR nanoparticles have a porous structure with a BET surface area of around 99.4 m(2) g(-1), an average pore size of about 6.14 nm and a pore volume of 0.47 cm(3) g(-1). Besides, the prepared restricted accessed Fe3O4@SiO2@PAR showed good size exclusion properties toward proteins, providing application potential for the direct analysis of biological samples. Based on this, a novel method of restricted accessed magnetic solid phase extraction (MSPE) combined with inductively coupled plasma-mass spectrometry (ICP-MS) was developed for the direct determination of trace metal ions in human fluids. The parameters affecting the extraction of the target metals by MSPE were studied and the optimized conditions were established. Under the optimum conditions, the adsorption capacity of Cr(III), Cd(II), La(III), Nd(III) and Pb(II) on the as-prepared restricted accessed Fe3O4@SiO2@PAR was 62.9, 56.6, 33.7, 36.9 and 43.3 mg g(-1), respectively. With an enrichment factor of 30, the limits of detection for Cr(III), Cd(II), La(III), Nd(III) and Pb(II) were as low as 11.9, 0.8, 0.7, 1.6 and 4.1 ng L(-1), and the relative standard deviations were 7.6, 8.7, 8.4, 8.1 and 5.0 (C(Cr, Pb) = 0.05 μg L(-1), C(Cd, La) = 0.005 μg L(-1), C(Nd) = 0.01 μg L(-1), n = 7), respectively. The developed method was successfully applied for the direct analysis of free metal ions in human urine and serum samples, and has the advantages of good anti-interference ability, high sensitivity and exhibits great application potential in the direct analysis of trace metals in biological fluids.

Towards point of care testing for C. difficile infection by volatile profiling, using the combination of a short multi-capillary gas chromatography column with metal oxide sensor detection

Rapid volatile profiling of stool sample headspace was achieved using a combination of short multi-capillary chromatography column (SMCC), highly sensitive heated metal oxide semiconductor sensor and artificial neural network software. For direct analysis of biological samples this prototype offers alternatives to conventional gas chromatography (GC) detectors and electronic nose technology. The performance was compared to an identical instrument incorporating a long single capillary column (LSCC). The ability of the prototypes to separate complex mixtures was assessed using gas standards and homogenized in house ‘standard’ stool samples, with both capable of detecting more than 24 peaks per sample. The elution time was considerably faster with the SMCC resulting in a run time of 10 min compared to 30 min for the LSCC. The diagnostic potential of the prototypes was assessed using 50 C. difficile positive and 50 negative samples. The prototypes demonstrated similar capability of discriminating between positive and negative samples with sensitivity and specificity of 85% and 80% respectively. C. difficile is an important cause of hospital acquired diarrhoea, with significant morbidity and mortality around the world. A device capable of rapidly diagnosing the disease at the point of care would reduce cases, deaths and financial burden.

Restricted access chiral stationary phase synthesized via reversible addition-fragmentation chain-transfer polymerization for direct analysis of biological samples by high performance liquid chromatography

Novel hydrophilic microparticles containing β-cyclodextrin (β-CD) were prepared via one-pot synthesis using reversible addition-fragmentation chain-transfer (RAFT) precipitation polymerization, a “controlled/living” radical polymerization technique. The polymerization was initiated by hydrophilic macromolecular chain-transfer agent [poly(2-hydroxyethyl methacrylate), PHEMA]. The hydrophilic PHEMA on the surface of microparticles can well improve their surface hydrophilicity and lead to their biological compatibility. As chiral restricted access material (RAM), the hydrophilic microparticles can be used for determination of enantiomers in biological samples with direct injection via HPLC analysis.

Analysis and Interpretation of Imaging Mass Spectrometry Data by Clustering Mass-to-Charge Images According to Their Spatial Similarity

Imaging mass spectrometry (imaging MS) has emerged in the past decade as a label-free, spatially resolved, and multipurpose bioanalytical technique for direct analysis of biological samples from animal tissue, plant tissue, biofilms, and polymer films. Imaging MS has been successfully incorporated into many biomedical pipelines where it is usually applied in the so-called untargeted mode-capturing spatial localization of a multitude of ions from a wide mass range.3 An imaging MS data set usually comprises thousands of spectra and tens to hundreds of thousands of mass-to-charge (m/z) images and can be as large as several gigabytes. Unsupervised analysis of an imaging MS data set aims at finding hidden structures in the data with no a priori information used and is often exploited as the first step of imaging MS data analysis. We propose a novel, easy-to-use and easy-to-implement approach to answer one of the key questions of unsupervised analysis of imaging MS data: what do all m/z images look like? The key idea of the approach is to cluster all m/z images according to their spatial similarity so that each cluster contains spatially similar m/z images. We propose a visualization of both spatial and spectral information obtained using clustering that provides an easy way to understand what all m/z images look like. We evaluated the proposed approach on matrix-assisted laser desorption ionization imaging MS data sets of a rat brain coronal section and human larynx carcinoma and discussed several scenarios of data analysis.

Testing for presence of known and unknown molecules in imaging mass spectrometry

Imaging mass spectrometry has emerged in the past decade as a label-free, spatially resolved and multi-purpose bioanalytical technique for direct analysis of biological samples. However, solving two everyday data analysis problems still requires expert judgment: (i) the detection of unknown molecules and (ii) the testing for presence of known molecules. We developed a measure of spatial chaos of a molecular image corresponding to a mass-to-charge value, which is a proxy for the molecular presence, and developed methods solving considered problems. The statistical evaluation was performed on a dataset from a rat brain section with test sets of molecular images selected by an expert. The measure of spatial chaos has shown high agreement with expert judges. The method for detection of unknown molecules allowed us to find structured molecular images corresponding to spectral peaks of any low intensity. The test for presence applied to a list of endogenous peptides ranked them according to the proposed measure of their presence in the sample. The source code and test sets of mass-to-charge images are available at http://www.math.uni-bremen.de/∼theodore. Supplementary materials are available at Bioinformatics online. theodore@uni-bremen.de.

Ambient laser ablation sampling for capillary electrophoresis mass spectrometry

Ambient laser ablation with mass spectrometric detection is a powerful method for direct analysis of biological samples in their native environment. Capillary electrophoresis (CE) can separate complex mixtures of biological molecules prior to mass spectrometry (MS) analysis and an ambient sampling interface for CE/MS will allow the detection of minor components. An infrared (IR) laser ablated and transferred sample materials under ambient conditions for direct loading onto the CE separation column. Samples were deposited on a transparent target and ablated in transmission geometry using a pulsed mid-IR laser. The ablated materials were captured in the exposed sampling solvent and then loaded into a capillary by electrokinetic injection for separation and analysis by electrospray ionization (ESI)-MS. The system was tested using mixtures of peptide and protein standards. It is estimated that tens of fmol of material was transferred from the ablation target for injection into the CE system and the theoretical plate number was between 1000 and 3000. A novel interface for ambient sampling to CE/MS was developed. The interface is generally applicable and has potential utility for mass spectrometry imaging as well as the loading of microfluidic devices from untreated ambient samples.

MRI-compatible pipeline for three-dimensional MALDI imaging mass spectrometry using PAXgene fixation

MALDI imaging mass spectrometry (MALDI-imaging) has emerged as a spatially-resolved label-free bioanalytical technique for direct analysis of biological samples and was recently introduced for analysis of 3D tissue specimens. We present a new experimental and computational pipeline for molecular analysis of tissue specimens which integrates 3D MALDI-imaging, magnetic resonance imaging (MRI), and histological staining and microscopy, and evaluate the pipeline by applying it to analysis of a mouse kidney. To ensure sample integrity and reproducible sectioning, we utilized the PAXgene fixation and paraffin embedding and proved its compatibility with MRI. Altogether, 122 serial sections of the kidney were analyzed using MALDI-imaging, resulting in a 3D dataset of 200GB comprised of 2million spectra. We show that elastic image registration better compensates for local distortions of tissue sections. The computational analysis of 3D MALDI-imaging data was performed using our spatial segmentation pipeline which determines regions of distinct molecular composition and finds m/z-values co-localized with these regions. For facilitated interpretation of 3D distribution of ions, we evaluated isosurfaces providing simplified visualization. We present the data in a multimodal fashion combining 3D MALDI-imaging with the MRI volume rendering and with light microscopic images of histologically stained sections. Our novel experimental and computational pipeline for 3D MALDI-imaging can be applied to address clinical questions such as proteomic analysis of the tumor morphologic heterogeneity. Examining the protein distribution as well as the drug distribution throughout an entire tumor using our pipeline will facilitate understanding of the molecular mechanisms of carcinogenesis.

Cluster TOF‐SIMS imaging: A new light for in situ metabolomics?

The advent of metal cluster as a primary ion source in the late 1980s, made it feasible to probe surfaces for complex organic structures due to a reduced in-source fragmentation, and opened the door to the direct analysis of biological samples. Despite the mass range measurable by TOF-secondary ion MS (SIMS) still being rather limited, the information obtained from cells and tissues comes together with the technical innovations introduced in the last decade. In this article, we give a brief overview of the technique itself and make some emphasis on the advances in the last three years in the analysis of biological surfaces, particularly those with direct implication in the biomedical field; reviewing what kind of information this instrumentation will add to current tool in pathology.

Infrared Mass Spectrometric Imaging below the Diffraction Limit

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)1 is an established technique for the analysis of biological macromolecules. Its relative insensitivity to pollutants makes MALDI-MS very suitable for the direct analysis of biological samples. As such, it has facilitated great advances in the field of biomolecular imaging mass spectrometry. Traditionally, MALDI-MS imaging is performed in a scanning microprobe methodology.(2-4) However, in a recent study we have demonstrated an alternative methodology; the so-called microscope mode,(5) where the requirement for a highly focused ionization beam is removed. Spatial details from within the desorption area are conserved during the flight of the ions through the mass analyzer, and a magnified ion image is projected onto a 2D-detector. In this paper, we demonstrate how imaging mass spectrometry benefits from the microscope mode approach. For the first time, high-lateral resolution ion images were recorded using infrared MALDI at 2.94 microm wavelength. The ion optical resolution achieved was well below the theoretical limit of (light-) diffraction for the setup used, which is impossible to achieve in the conventional scanning microprobe approach.

Direct Analysis of Biological Samples by Total Reflection X‐Ray Fluorescence

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis and characterization of polymer-coated mixed functional stationary phases with several different hydrophobic groups for direct analysis of biological samples by liquid chromatography

Three types of polymer-coated mixed-functional (PCMF) silica column-packing materials for the direct analysis of biological liquids have been synthesized. These packing materials all have polyoxyethylene groups as their hydrophilic part but differ in their hydrophobic part (methyl groups for Me-POE, phenyl groups for Ph-POE, and octyl groups for Oc-POE). Retention characteristics with respect to several drug molecules, and protein recovery, were studied for each of the three types with different amounts of hydrophobic groups attached. Of the PCMFs prepared, Oc-POE showed the greatest overall retention, and Me-POE displayed the greatest protein recovery.

Comparison of standard materials for the establishment of calibration curves in solid sampling graphite furnace atomic absorption spectrometry. Direct determination of copper in powdered biological samples

The selection of standard materials for the establishment of calibration curves is one of the most important problems in the direct analysis of biological samples by GFAAS. Three kinds of standard materials, NBS tomato leaves, coprecipitates with magnesium oxinate and Ni/DMG/PAN were investigated. It was found that it is possible to use both the NBS-SRM and the synthetic reference material prepared by coprecipitation with magnesium oxinate as standard materials for the direct determination of copper in several biological samples issued from NBS and NIES. However, too low results are obtained when the synthetic reference material prepared by coprecipitation with Ni/DMG/PAN is used. In order to clarify this, the effects of nickel and magnesium were examined and the role of magnesium was discussed.

The structural identification of drug metabolites using thermospray liquid chromatography/mass spectrometry.

The structural identification of drug metabolites has been carried out using thermospray liquid chromatography/mass spectrometry (LC/MS). It has allowed the direct analysis of biological samples, in this case in vitro hepatocyte incubations, with the minimum of sample preparation. The technique also provided molecular weight information on several conjugates including glucuronides, a glutathione conjugate and one unidentified conjugate. A number of minor metabolites were also successfully identified using this method. The examples discussed in this paper illustrate the value of LC/MS in identifying unknown drug metabolites covering a wide polarity range in a complex biological mixture. However, this would not have been possible, if the interface had been unable to handle gradient separations.