Quantitative Matrix-assisted Laser Research Articles

Tracking Drugs and Lipids: Quantitative Mass Spectrometry Imaging of Liposomal Doxorubicin Delivery and Bilayer Fate in Three-Dimensional Tumor Models.

Targeted therapy to the tumor would greatly advance precision medicine. Many drug delivery vehicles have emerged, but liposomes are cited as the most successful to date. Recent efforts to develop liposomal drug delivery systems focus on drug distribution in tissues and ignore liposomal fate. In this study, we developed a novel method to elucidate both drug and liposomal bilayer distribution in a three-dimensional cell culture model using quantitative matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI qMSI) alongside fluorescence microscopy. Imaging liposomal distribution in a cell culture model is challenging, as lipids forming the bilayer are endogenous to the model system. To resolve this issue, we functionalized the bilayer by chemically cross-linking a fluorescent tag to the alkyne-containing lipid hexynoyl phosphoethanolamine (HPE). We synthesized liposomes incorporating the tagged HPE lipid and encapsulated within them doxorubicin, yielding a theranostic liposome capable of both drug delivery and monitoring liposomal uptake. We employed an "in-tissue" MALDI qMSI approach to generate a calibration curve with R2 = 0.9687, allowing for quantification of doxorubicin within spheroid sections at multiple time points. After 72 h of treatment with the theranostic liposomes, full doxorubicin penetration was observed. The metabolites doxorubicinone and 7-deoxydoxorubicinone were also detected after 48 h. Modification of the bilayer allowed for fluorescence microscopy tracking of liposomes, while MALDI MSI simultaneously permitted the imaging of drugs and metabolites. While we demonstrated the utility of our method with doxorubicin, this system could be applied to examine the uptake, release, and metabolism of many other liposome-encapsulated drugs.

2-Hydrazinoterephthalic Acid as a Novel Negative-Ion Matrix-Assisted Laser Desorption/Ionization Matrix for Qualitative and Quantitative Matrix-Assisted Laser Desorption/Ionization–Mass Spectrometry Analysis of N-Glycans in Peach Allergy Research

Glycans recently attracted considerable attention as the proposal of cross-reactive carbohydrate determinants for food allergy. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is powerful in analyzing biomolecules, while its applications in glycans are still challenging. Herein, a novel reactive matrix-assisted laser desorption/ionization (MALDI) matrix, 2-hydrazinoterephthalic acid, was rationally designed and synthesized. It provides uniform co-crystallization with glycans and only produces deprotonated ions with high intensities in the negative-ion mode. In combination with sinapic acid, a rapid and high-throughput method was established for on-target analysis of glycans with a superior limit of detection at the femtomole level and a good linearity (R2 > 0.999). Furthermore, the established method was successfully applied to quantify N-glycans in different cultivars and tissues of peach [Prunus persica (L.) Batsch]. Our work suggests the potential role of N-glycans as biomarkers for food-borne allergy and lays a methodological foundation for the elucidation of the possible relationship between carbohydrate epitopes and food allergy.

Prevalence of monoclonal gammopathies and clinical outcomes in a high-risk US population screened by mass spectrometry: a multicentre cohort study

Prevalence estimates for monoclonal gammopathy of undetermined significance (MGUS) are based on predominantly White study populations screened by serum protein electrophoresis supplemented with immunofixation electrophoresis. A prevalence of 3% is reported for MGUS in the general population of European ancestry aged 50 years or older. MGUS prevalence is two times higher in individuals of African descent or with a family history of conditions related to multiple myeloma. We aimed to evaluate the prevalence and clinical implications of monoclonal gammopathies in a high-risk US population screened by quantitative mass spectrometry. We used quantitative matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and EXENT-iQ software to screen for and quantify monoclonal gammopathies in serum from 7622 individuals who consented to the PROMISE screening study between Feb 26, 2019, and Nov 4, 2021, and the Mass General Brigham Biobank (MGBB) between July 28, 2010, and July 1, 2021. M-protein concentrations at the monoclonal gammopathy of indeterminate potential (MGIP) level were confirmed by liquid chromatography mass spectrometry testing. 6305 (83%; 2211 from PROMISE, 4094 from MGBB) of 7622 participants in the cohorts were at high risk for developing a monoclonal gammopathy on the basis of Black race or a family history of haematological malignancies and fell within the eligible high-risk age range (30 years or older for PROMISE cohort and 18 years or older for MGBB cohort); those over 18 years were also eligible if they had two or more family members with a blood cancer (PROMISE cohort). Participants with a plasma cell malignancy diagnosed before screening were excluded. Longitudinal clinical data were available for MGBB participants with a median follow-up time from serum sample screening of 4·5 years (IQR 2·4-6·7). The PROMISE study is registered with ClinicalTrials.gov, NCT03689595. The median age at time of screening was 56·0 years (IQR 46·8-64·1). 5013 (66%) of 7622 participants were female, 2570 (34%) male, and 39 (<1%) unknown. 2439 (32%) self-identified as Black, 4986 (65%) as White, 119 (2%) as other, and 78 (1%) unknown. Using serum protein electrophoresis with immunofixation electrophoresis, the MGUS prevalence was 6% (101 of 1714) in high-risk individuals aged 50 years or older. Using mass spectrometry, we observed a total prevalence of monoclonal gammopathies of 43% (1788 of 4207) in this group. We termed monoclonal gammopathies below the clinical immunofixation electrophoresis detection level (<0·2 g/L) MGIPs, to differentiate them from those with higher concentrations, termed mass-spectrometry MGUS, which had a 13% (592 of 4207) prevalence by mass spectrometry in high-risk individuals aged 50 years or older. MGIP was predominantly of immunoglobulin M isotype, and its prevalence increased with age (19% [488 of 2564] for individuals aged <50 years, 29% [1464 of 5058] for those aged ≥50 years, and 37% [347 of 946] for those aged ≥70 years). Mass-spectrometry MGUS prevalence increased with age (5% [127 of 2564] for individuals aged <50 years, 13% [678 of 5058] for those aged ≥50 years, and 18% [173 of 946] for those aged ≥70 years) and was higher in men (314 [12%] of 2570) compared with women (485 [10%] 5013; p=0·0002), whereas MGIP prevalence did not differ significantly by gender. In those aged 50 years or older, the prevalence of mass spectrometry was significantly higher in Black participants (224 [17%] of 1356) compared with the controls (p=0·0012) but not in those with family history (368 [13%] of 2851) compared with the controls (p=0·1008). Screen-detected monoclonal gammopathies correlated with increased all-cause mortality in MGBB participants (hazard ratio 1·55, 95% CI 1·16-2·08; p=0·0035). All monoclonal gammopathies were associated with an increased likelihood of comorbidities, including myocardial infarction (odds ratio 1·60, 95% CI 1·26-2·02; p=0·00016 for MGIP-high and 1·39, 1·07-1·80; p=0·015 for mass-spectrometry MGUS). We detected a high prevalence of monoclonal gammopathies, including age-associated MGIP, and made more precise estimates of mass-spectrometry MGUS compared with conventional gel-based methods. The use of mass spectrometry also highlighted the potential hidden clinical significance of MGIP. Our study suggests the association of monoclonal gammopathies with a variety of clinical phenotypes and decreased overall survival. Stand Up To Cancer Dream Team, the Multiple Myeloma Research Foundation, and National Institutes of Health.

Stochastic dynamic quantitative and 3D structural matrix assisted laser desorption/ionization mass spectrometric analyses of mixture of nucleosides

The paper tackles issue of solid-state 3D structural analysis by means of matrix assisted laser desorption/ionization mass spectrometry with a view of our innovative stochastic dynamic new simplistic formula D”SD = 2.6388.10−17.(<I2>–<I>2). It represents a universal law, which is obeyed by temporal behavior of experimental variable intensity of any analyte peak of ion, measured in solution under soft-ionization electrospray ionization, collision induced dissociation and atmospheric pressure chemical ionization conditions, respectively. The equation connects between so-called stochastic dynamic diffusion parameter, D”SD, and variable intensity, I, determined per any span of scan time of a measurement. So far, it has been proven empirically, that D”SD parameters of MS ions reflect exactly analyte concentration in solution — a claim independently verified via chromatography — and provide exact information about 3D molecular and electronic structure of molecular species in liquid phase. Herein, we urge that this simplistic formula is a fundamental law also valid to MALDI-MS phenomena; thus, providing exact quantitative and 3D molecular structural information about analytes in solid-state; or, mass spectrometric phenomena in condensed phases are governed by one and the same stochastic dynamic law, which is applicable both to quantitative and 3D analyses of analytes. The latter statement underlines a crucial advantage of the functional relation and emphasizes on incapability of other known fundamental MS theories and equation to deal with simultaneous exact quantitative and 3D structural analyses in the two condensed phases. A body of new empirical evidence is directed to favor the view of application of the model equation to MALDI-MS processes, examining guanosine (1) in mixture of N(2),N(2)-dimethyl guanosine, quantitatively and 3D structurally, with respect to different experimental conditions without, and involving different sample matrix components. The ultra-high accuracy mass spectrometric outcomes are treated by chemomerics. The same is true for utilizing high accuracy quantum chemical static and molecular dynamics in order to correlate experimental mass spectrometric with theoretical quantum chemical data on 3D molecular and electronic structures of nucleosides, their fragment species and energetics in gas and condensed phases. There are detailed subtle electronic effects on various tautomers, ionization processes, radical-ion formation MS reactions, chemical rearrangement, charge and (doubly) proton transfer effects; if any.

Determination of Uropathogenic Escherichia coli in Urine by an Immunobiosensor Based Upon Antigen-Antibody Biorecognition with Fluorescence Detection and Bead-Injection Analysis

Uroinfections, which are mainly caused by pathogenic bacteria, especially uro-pathogenic E. coli (UPEC), are among the major health problems in the modern world. The current gold standard for the identification of uroinfection causing pathogens in urine is microbiological cultivation, but uro-pathogens are also determined with quantitative polymerase chain reaction (qPCR) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. However, it takes several hours or days to obtain the results. A promising option for rapid identification of pathogens is the application of biosensors. The aim of the current study was to develop a biosensor for the fast detection of UPEC in urine samples allowing achieving a limit of detection below 103 CFU/mL, essential for the diagnostics of recurrent uroinfections and related diseases. First, the pathogens were attached onto a single use column and specifically detected using E. coli antibodies conjugated with a fluorescence marker. The bead-injection analysis platform for fluidics provided E. coli limits of detection and quantification in 150 µL urine samples <3 and <5 cells/mL, respectively. The time of analysis was 17 min. The biosensor results were in good correlation with the results obtained with other methods, indicating that the complex urine matrix of UPEC patients did not affect the biosensor measurements.

Oriented Functionalization of Magnetic Beads with in Vivo Biotinylated Nanobodies for Rapid MALDI-TOF MS Ultrasensitive Quantitation of Microcystins in Biological Samples.

Here we present a new analytical method where immunoconcentration of the analyte is coupled to quantitative matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis allowing in minutes the identification and highly sensitive quantitation of microcystins (MCs) as model targets. The key element is a site-specific in vivo biotinylated nanobody of broad cross-reactivity with microcystins. The single biotin moiety at the C-terminus and the small size of the nanobody (15 kDa) enable its oriented and tightly packed immobilization on magnetic beads, providing a highly efficient capture of the toxin. The binding capacity of the bioadsorbent is partially loaded with an easily synthesized internal standard for MS quantitation. After capture, the beads are directly dispensed on the MALDI-TOF MS target enabling the identification and sensitive quantitation of the microcystin (MC) congeners. Since salts and contaminants are removed during the concentration step, no cleanup or other sample treatments are needed. The method was validated with a large number of water and serum samples with excellent precision and recovery at quantitation limits of 0.025 μg/L of MC.

Selection of Internal Standards for Quantitative Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis Based on Correlation Coefficients.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has shown its great success in the qualitative analysis of a wide range of organic and biological molecules. However, its application in quantitative analysis is still limited by the difficulty in the availability of isotope-labeled internal standards. The present work investigates the relationship between the correlation coefficient of the peak intensities of analyte and candidate internal standard ions and the linearity of possible quantitative analysis. Based on the two analyte examples, ciprofloxacin and substance P, the results show that the performance of the selected nonisotope-labeled internal standard is greatly related to the correlation coefficient. A high positive correlation coefficient (>0.7) between the ions of analyte and candidate standard can result in a good linearity (R2 > 0.98) and vice versa. The results provide a new way to select nonisotope-labeled internal standards for MALDI analysis and thus can be potentially applied in the rapid quantitative mass spectrometry.

Pharmacometabolic response to pirfenidone in pulmonary fibrosis detected by MALDI-FTICR-MSI.

Idiopathic pulmonary fibrosis (IPF) is a fatal condition that reduces life expectancy and shows a limited response to available therapies. Pirfenidone has been approved for treatment of IPF, but little is known about the distinct metabolic changes that occur in the lung upon pirfenidone administration.Here, we performed a proof-of-concept study using high-resolution quantitative matrix-assisted laser desorption/ionisation Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI) to simultaneously detect, visualise and quantify in situ endogenous and exogenous metabolites in lungs of mice subjected to experimental fibrosis and human patients with IPF, and to assess the effect of pirfenidone treatment on metabolite levels.Metabolic pathway analysis and endogenous metabolite quantification revealed that pirfenidone treatment restores redox imbalance and glycolysis in IPF tissues, and downregulates ascorbate and aldarate metabolism, thereby likely contributing to in situ modulation of collagen processing. As such, we detected specific alterations in metabolite pathways in fibrosis and, importantly, metabolic recalibration following pirfenidone treatment.Together, these results highlight the suitability of high-resolution MALDI-FTICR-MSI for deciphering the therapeutic effects of pirfenidone and provide a preliminary analysis of the metabolic changes that occur during pirfenidone treatment in vivo These data may therefore contribute to improvement of currently available therapies for IPF.

Multiplexed Quantitative MALDI MS Approach for Assessing Activity and Inhibition of Protein Kinases Based on Postenrichment Dephosphorylation of Phosphopeptides by Metal–Organic Framework-Templated Porous CeO2

Protein kinase is regarded as a potential target for anticancer therapeutics due to its relation to many diseases, in which more than one kinase participates in the cell signaling cascades. We herein demonstrate a multiplexed quantitative matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) approach to simultaneously assess the activity and inhibition of multiple protein kinases. In this design, substrate peptides phosphorylated by kinases of interest are specifically harvested by metal-organic framework (MOF)-templated porous CeO2 and consequently transformed to the dephosphorylated forms due to the phosphatase-like activity of CeO2, resulting in a unique quantitative MS signal with an enhanced intensity. Based on the peak area ratios of dephosphorylated variants of the phosphorylated product to respective deuterated internal standard, the activity and inhibition of each kinase can be independently profiled. In addition to the accurate characterization of protein kinase A (PKA) activity and inhibition induced by H-89, the multiplexing capability of the MS-based method allowed quantitative evaluation of the activity of Abl and Src, the two tightly associated kinases in the occurrence of chronic myeloid leukemia (CML) in a multiplexed format, exhibiting excellent orthogonality for the dual signal readout channels. Moreover, the inactivation of both Abl and Src by the inhibitor imatinib, dasatinib, and ponatinib was simultaneously traced, giving a whole picture of the competition behavior between the kinases for binding therapeutic molecules. This approach holds great promise in global investigation of kinase signal pathways and high-throughput screening of effective protein kinase inhibitors.

Miniaturized dispersive liquid-liquid microextraction and MALDI MS using ionic liquid matrices for the detection of bacterial communication molecules and virulence factors.

The identification and quantification of molecules involved in bacterial communication are major prerequisites for the understanding of interspecies interactions at the molecular level. We developed a procedure allowing the determination of 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and the virulence factor pyocyanin (PYO) formed by the Gram-negative bacterium Pseudomonas aeruginosa. The method is based on dispersive liquid-liquid microextraction from small supernatant volumes (below 10μL) followed by quantitative matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The use of ionic liquid matrix led to a lowered limit of detection for pyocyanin and, due to suppression of matrix background signals, easy to interpret mass spectra compared to crystalline matrices. Using an isotope-labeled pyocyanin standard synthesized in small-scale synthesis, quantitative analysis spanning approximately one order of magnitude (0.5 to 250fmol) was feasible. The method was successfully applied to the detection of the signaling molecules PQS and HHQ in cultures of P. aeruginosa strains isolated from sputum of cystic fibrosis patients and allowed a highly sensitive quantification of PYO from these cultures. Hence, the developed method bears the potential to be used for screening purposes in clinical settings and will help to decipher the molecular basis of bacterial communication. Graphical abstract Ionic liquid matrices for the detection and quantification of the toxin pyocyanin and other signaling molecules from P. aeruginosa by MALDI MS.

Mechanochemical Amorphization of α-Chitin and Conversion into Oligomers of N-Acetyl-d-glucosamine

Mechanochemical treatment offers great potential for environmentally sustainable processing of chitin within the context of biomass valorization. Using powder X-ray diffraction, we show that crystallinity can be reduced by 50% in 2 h in a controlled way using a ball mill. We correlate this crystallinity reduction with a decrease in interchain hydrogen bonding using infrared spectroscopy as a structural probe. Furthermore, our quantitative interpretation of the spectra reveal a decrease in glycosidic linkage content and retention of N-acetyl groups. The addition of a natural clay, kaolinite, in the ball mill leads to a significant increase in the solubility of the milled materials (up to 75.8% water-soluble products in 6 h, cf. 35.0% without kaolinite). The products of this process were characterized as oligomers of N-acetyl-d-glucosamine (chitin oligomers) with degrees of polymerization (DP) between 1 and 5 using a new quantitative matrix-assisted laser desorption-ionization (MALDI-ToF) mass spectrometric...

Direct and Indirect Photochemical Reactions in Viral RNA Measured with RT-qPCR and Mass Spectrometry.

RNA carries the genetic instructions for many viruses to replicate in their host cells. The photochemical reactions that take place in RNA and affect viral infectivity in natural and engineered environments, however, remain poorly understood. We exposed RNA oligomer segments from the genome of bacteriophage MS2 to UV254, simulated sunlight, and singlet oxygen (1O2) and analyzed the oligomer reaction kinetics with reverse transcription quantitative PCR (RT-qPCR) and quantitative matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Following UV254 exposure, quantitative MALDI-TOF-MS detected significantly more RNA modifications than did RT-qPCR, suggesting that certain chemical modifications in the RNA were not detected by the reverse transcriptase enzyme. In contrast, MALDI-TOF-MS tracked as much 1O2-induced RNA damage as RT-qPCR. After 5 h of simulated sunlight exposure (5100 J/m2 UVB and 1.2 × 105 J/m2 UVA), neither MALDI-TOF-MS nor RT-qPCR detected significant decreases in the oligomer concentrations. High-resolution electrospray ionization (ESI)-Orbitrap MS analyses identified pyrimidine photohydrates as the major UV254 products, which likely contributed to the discrepancy between the MS- and RT-qPCR-based results. Reactions between RNA oligomers and 1O2 resulted in an unidentified major product with a mass change of +6 Da. These results shed light on the photochemical reactions that take place in RNA and suggest that the analytical techniques used to detect RNA reactivity could bias the observed reaction kinetics.

Multiple Time-of-Flight/Time-of-Flight Events in a Single Laser Shot for Improved Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry Quantification.

Quantitative matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) approaches have historically suffered from poor accuracy and precision mainly due to the nonuniform distribution of matrix and analyte across the target surface, matrix interferences, and ionization suppression. Tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity as well as improve signal-to-noise ratios by eliminating interferences from chemical noise, alleviating some concerns about dynamic range. However, conventional MALDI TOF/TOF modalities typically only scan for a single MS/MS event per laser shot, and multiplex assays require sequential analyses. We describe here new methodology that allows for multiple TOF/TOF fragmentation events to be performed in a single laser shot. This technology allows the reference of analyte intensity to that of the internal standard in each laser shot, even when the analyte and internal standard are quite disparate in m/z, thereby improving quantification while maintaining chemical specificity and duty cycle. In the quantitative analysis of the drug enalapril in pooled human plasma with ramipril as an internal standard, a greater than 4-fold improvement in relative standard deviation (<10%) was observed as well as improved coefficients of determination (R2) and accuracy (>85% quality controls). Using this approach we have also performed simultaneous quantitative analysis of three drugs (promethazine, enalapril, and verapamil) using deuterated analogues of these drugs as internal standards.

Matrix normalized MALDI-TOF quantification of a fluorotelomer-based acrylate polymer.

The degradation of fluorotelomer-based acrylate polymers (FTACPs) has been hypothesized to serve as a source of the environmental contaminants, perfluoroalkyl carboxylates (PFCAs). Studies have relied on indirect measurement of presumed degradation products to evaluate the environmental fate of FTACPs; however, this approach leaves a degree of uncertainty. The present study describes the development of a quantitative matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry method as the first direct analysis method for FTACPs. The model FTACP used in this study was poly(8:2 FTAC-co-HDA), a copolymer of 8:2 fluorotelomer acrylate (8:2 FTAC) and hexadecyl acrylate (HDA). Instead of relying on an internal standard polymer, the intensities of 40 poly(8:2 FTAC-co-HDA) signals (911-4612 Da) were normalized to the signal intensity of a matrix-sodium cluster (659 Da). We termed this value the normalized polymer response (P(N)). By using the same dithranol solution for the sample preparation of poly(8:2 FTAC-co-HDA) standards, calibration curves with coefficient of determinations (R(2)) typically >0.98 were produced. When poly(8:2 FTAC-co-HDA) samples were prepared with the same dithranol solution as the poly(8:2 FTAC-co-HDA) standards, quantification to within 25% of the theoretical concentration was achieved. This approach minimized the sample-to-sample variability that typically plagues MALDI-TOF, and is the first method developed to directly quantify FTACPs.

Simple quantitation for potential serum disease biomarker peptides, primarily identified by a peptidomics approach in the serum with hypertensive disorders of pregnancy.

We previously reported peptide candidates of disease biomarkers for pregnancy-induced hypertension syndrome using a novel peptidomic analytical method, BLOTCHIP®-MS. The aim of this study was to establish a sandwich enzyme-linked immunosorbent assay system for quantitation of such peptides and to validate their usefulness as disease biomarkers of pregnancy-induced hypertension syndrome including gestational hypertension/pre-eclampsia. We focused on three peptide fragments, kininogen-1439-456 (PDA039), kininogen-1438-456 (PDA044) and cysteinyl α2-HS-glycoprotein341-367 (PDA071). Using polyclonal antibodies specific for each peptide, suitable conditions for the sandwich enzyme-linked immunosorbent assay system were investigated. The quantitative enzyme-linked immunosorbent assay values were confirmed by quantitative matrix assisted laser desorption/ionization time-of-flight MS analyses. Using the established enzyme-linked immunosorbent assay systems, serum samples from gestational hypertension/pre-eclampsia patients and paired serum samples from healthy pregnant females were analysed. The optimum sandwich enzyme-linked immunosorbent assay conditions for PDA039/044 quantitation were developed. Quantitation of PDA071 by enzyme-linked immunosorbent assay failed, presumably due to issues with polyclonal antibody specificity for the native peptide. Bland-Altman plots showed a satisfactory correlation between the serum PDA039/044 concentration by enzyme-linked immunosorbent assay and that by quantitative MS analysis. Although the PDA044 concentration showed no significant change during pregnancy, including gestational hypertension/pre-eclampsia patients, the serum PDA039 concentration was significantly increased (P < 0.0001) in the patients. The simple quantitation technology for PDA039 by enzyme-linked immunosorbent assay was established for the first time. PDA039 confirmed its clinical utility as a disease biomarker for gestational hypertension/pre-eclampsia by the enzyme-linked immunosorbent assay system using clinical samples. The information provided from the present study would be a new valuable addition in the field of gestational hypertension/pre-eclampsia research.

Sinapinic acid-directed synthesis of gold nanoclusters and their application to quantitative matrix-assisted laser desorption/ionization mass spectrometry

Core etching of gold nanoparticles (AuNPs) into smaller-sized clusters is a classic method for fabricating gold nanoclusters (AuNCs). The top down-based synthesis of AuNCs includes two steps: (i) reducing the Au(3+) precursor solution to generate AuNPs in the presence of protecting ligands and (ii) core etching of the formed AuNPs into the AuNCs via ligand exchange. For the first time, this paper describes a one-step approach for preparing AuNCs using a top down approach. The sinapinic acid (SA)-induced formation of the AuNCs involved a three-step reaction process. First, large AuNPs (>200 nm) were quickly formed after mixing SA and the Au(3+) precursor solution. Second, excess SA molecules self-assembled on the NP surface, and large AuNPs were etched to small AuNPs via electrostatic repulsion between the neighboring SA molecules. Finally, SA-induced core etching of the AuNPs resulted in the formation of the AuNCs within 70 min. Furthermore, we showed that the presence of the AuNCs in SA was capable of suppressing crystal growth and eliminating the coffee-ring effect. Thus, proteins can be successfully quantified using the SA-AuNCs as matrices for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Compared with using SA as matrices, the SA-AuNCs offered substantial advantages for improving shot-to-shot reproducibility and enhancing the ionization efficiency of proteins.

Self-Aliquoting Microarray Plates for Accurate Quantitative Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a fast analysis tool employed for the detection of a broad range of analytes. However, MALDI-MS has a reputation of not being suitable for quantitative analysis. Inhomogeneous analyte/matrix co-crystallization, spot-to-spot inhomogeneity, as well as a typically low number of replicates are the main contributing factors. Here, we present a novel MALDI sample target for quantitative MALDI-MS applications, which addresses the limitations mentioned above. The platform is based on the recently developed microarray for mass spectrometry (MAMS) technology and contains parallel lanes of hydrophilic reservoirs. Samples are not pipetted manually but deposited by dragging one or several sample droplets with a metal sliding device along these lanes. Sample is rapidly and automatically aliquoted into the sample spots due to the interplay of hydrophilic/hydrophobic interactions. With a few microliters of sample, it is possible to aliquot up to 40 replicates within seconds, each aliquot containing just 10 nL. The analyte droplet dries immediately and homogeneously, and consumption of the whole spot during MALDI-MS analysis is typically accomplished within few seconds. We evaluated these sample targets with respect to their suitability for use with different samples and matrices. Furthermore, we tested their application for generating calibration curves of standard peptides with α-cyano-4-hdydroxycinnamic acid as a matrix. For angiotensin II and [Glu(1)]-fibrinopeptide B we achieved coefficients of determination (r(2)) greater than 0.99 without the use of internal standards.

Subtle Differences in Virus Composition Affect Disinfection Kinetics and Mechanisms

Viral disinfection kinetics have been studied in depth, but the molecular-level inactivation mechanisms are not understood. Consequently, it is difficult to predict the disinfection behavior of nonculturable viruses, even when related, culturable viruses are available. The objective of this work was to determine how small differences in the composition of the viral genome and proteins impact disinfection. To this end, we investigated the inactivation of three related bacteriophages (MS2, fr, and GA) by UV254, singlet oxygen ((1)O2), free chlorine (FC), and chlorine dioxide (ClO2). Genome damage was quantified by PCR, and protein damage was assessed by quantitative matrix-assisted laser desorption ionization (MALDI) mass spectrometry. ClO2 caused great variability in the inactivation kinetics between viruses and was the only treatment that did not induce genome damage. The inactivation kinetics were similar for all viruses when treated with disinfectants possessing a genome-damaging component (FC, (1)O2, and UV254). On the protein level, UV254 subtly damaged MS2 and fr capsid proteins, whereas GA's capsid remained intact. (1)O2 oxidized a methionine residue in MS2 but did not affect the other two viruses. In contrast, FC and ClO2 rapidly degraded the capsid proteins of all three viruses. Protein composition alone could not explain the observed degradation trends; instead, molecular dynamics simulations indicated that degradation is dictated by the solvent-accessible surface area of individual amino acids. Finally, despite the similarities of the three viruses investigated, their mode of inactivation by a single disinfectant varied. This explains why closely related viruses can exhibit drastically different inactivation kinetics.

Quantitative Matrix-assisted Laser Desorption/ionization Mass Spectrometry of Pyrene-derivatized Glycopeptides for Investigation of Mammalian Cell Glycomics

Glycan is one of the major information-rich biomolecules. Alterations in N-glycans can be associated with many diseases including cancer, and are often observed in the serum of affected patients. As an example, prostate-specific antigen (PSA) is a glycoprotein secreted by prostatic epithelial cells. The serum PSA assay is widely used for detection of prostate cancer (PCa). Detection of altered PSA glycan is regarded as a precise diagnosis. However, the limited amount of serum PSA available makes it difficult to determine detailed glycan structures, as the PSA level in serum is very low. Mass spectrometry (MS) is a powerful tool for analyzing glycan structures. We have recently established a highly sensitive MS for both glycans and glycopeptides by pyrene derivatization. Matrix-assisted laser desorption/ionizationquadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectra of glycans derivatized with pyrene butanoic acid hydrazide (PBH) were compared with those of pyridylaminated (PA) glycans. Each derivatized glycan was prepared from the same amount of commercial PSA. PBH-labeled PSA glycans showed higher signal intensity with less fragmentation compared with PA-glycans, and gave spectra with high s/n ratio (in both positive- and negative-ion modes). For derivatization of the glycopeptides, pyrenyl diazomethane (PDAM) was used. The glycopeptide signals were greatly enhanced by this derivatization. PDAM-glycopeptides prepared from about 10 ng of PSA could be determined. In this study, we demonstrated that MS analysis using pyrene derivatization provides higher sensitivity and stability for both glycans and glycopeptides, compared with HPLC and lectin affinity chromatography; furthermore comparable results were obtained to those seen with the other methods. We therefore conclude that our method is useful for investigation of mammalian cell glycomics.

Minocycline Effects on the Cerebrospinal Fluid Proteome of Experimental Autoimmune Encephalomyelitis Rats

To identify response biomarkers for pharmaceutical treatment of multiple sclerosis, we induced experimental autoimmune encephalomyelitis (EAE) in rats and treated symptomatic animals with minocycline. Cerebrospinal fluid (CSF) samples were collected 14 days after EAE induction at the peak of neurological symptoms, and proteomics analysis was performed using nano-LC-Orbitrap mass spectrometry. Additionally, the minocycline concentration in CSF was determined using quantitative matrix-assisted laser desorption/ionization-triple-quadrupole tandem mass spectrometry (MALDI-MS/MS) in the selected reaction monitoring (SRM) mode. Fifty percent of the minocycline-treated EAE animals did not show neurological symptoms on day 14 ("responders"), while the other half displayed neurological symptoms ("nonresponders"), indicating that minocycline delayed disease onset and attenuated disease severity in some, but not all, animals. Neither CSF nor plasma minocycline concentrations correlated with the onset of symptoms or disease severity. Analysis of the proteomics data resulted in a list of 20 differentially abundant proteins between the untreated animals and the responder group of animals. Two of these proteins, complement C3 and carboxypeptidase B2, were validated by quantitative LC-MS/MS in the SRM mode. Differences in the CSF proteome between untreated EAE animals and minocycline-treated responders were similar to the differences between minocycline-treated responders and nonresponders (70% overlap). Six proteins that remained unchanged in the minocycline-treated animals but were elevated in untreated EAE animals may be related to the mechanism of action of minocycline.