Mass Spectrometric Monitoring Research Articles

Water Microdroplets Surrounded by Alcohol Vapor Cause Spontaneous Oxidation of Alcohols to Organic Peroxides.

Using real-time mass spectrometric (MS) monitoring, we demonstrate one-step, catalyst-free spontaneous oxidation of various alcohols (ROH) to key reactive intermediates for the formation of ROO- compounds on the surface of water microdroplets surrounded by alcohol vapor, carried out under ambient conditions. These organic peroxides (POs) can act as important secondary organic aerosols (SOA). We used hydrogen-deuterium exchange by spraying D2O instead of H2O to learn about the reaction mechanism, and the results demonstrate the crucial role of the water-air interface in microdroplet chemistry. We find that the formation of POs relies on electron transfer occurring at the microdroplet interface, which generates hydrogen atoms and hydroxyl radicals that lead to a cascade of radical reactions. This electron transfer is believed to be driven by two factors: (1) the emergence of a strong electrostatic potential on the microdroplet's surface; and (2) the partial solvation of ions at the interface. Mass spectra reveal that the formation of POs is dependent on the alcohol structure, with tertiary alcohols showing a higher tendency to form organic peroxides than secondary alcohols, which in turn are more reactive than primary alcohols.

New thermal decomposition pathway for TATB

Understanding the thermal decomposition behavior of TATB (1,3,5-triamino-2,4,6-trinitrobenzene) is a major focus in energetic materials research because of safety issues. Previous research and modelling efforts have suggested benzo-monofurazan condensation producing H2O is the initiating decomposition step. However, early evolving CO2 (m/z 44) along with H2O (m/z 18) evolution have been observed by mass spectrometric monitoring of head-space gases in both constant heating rate and isothermal decomposition studies. The source of the CO2 has not been explained, until now. With the recent successful synthesis of 13C6-TATB (13C incorporated into the benzene ring), the same experiments have been used to show the source of the CO2 is the early breakdown of the TATB ring, not adventitious C from impurities and/or adsorbed CO2. A shift in mass m/z 44 (CO2) to m/z 45 is observed throughout the decomposition process indicating the isotopically labeled 13C ring breakdown occurs at the onset of thermal decomposition along with furazan formation. Partially labeled (N18O2)3-TATB confirms at least some of the oxygen comes from the nitro-groups. This finding has a significant bearing on decomposition computational models for prediction of energy release and deflagration to detonation transitions, with respect to conditions which currently do not recognize this oxidation step.

Mass spectrometric monitoring of ligand-bridged hot electron transfer and anaerobic oxidization on auto renewable droplet-based plasmonic nanoreactors under visible light illumination

The light induced hot-electron on plasmonic nanostructures has been recognized as a breakthrough discovery for photovoltaic and photocatalytic applications. With mass spectrometry, we demonstrate the dynamics of hot electron transfers of anaerobic oxidization reactions on Au decorated TiO2 plasmonic nanoparticles, which were coated on the inner surface of a flask. Those nanoparticles were covered by continuously renewed liquid droplets of solvent and reactants that were transported through a Venturi jet mixer with auto-spray. In addition to intensive mass transfer in such droplet-based nanoreactors, as well as strong adsorption of reactants and rapid desorption of products on materials surfaces, the localized surface plasmon resonance (LSPR) excitation upon visible light illumination, by which accumulated energies of plasmons are transferred to electrons in the conduction band of the material, attributes to the efficient photocatalytic transformation. Mass spectrometric detection of intermediate radical anions and negative ions with stable isotope labeling unambiguously identifies that highly energetic hot electrons can escape from the plasmonic nanostructures, be collected by adsorbed molecules, and initiate bond cleavages. It was demonstrated that losses of two H atoms result in the anaerobic oxidization of each benzyl alcohol molecule to a benzyl aldehyde molecule in the absence of molecular oxygen with more than 90 % yields. The well recyclable plasmonic nanoreactors implicate the injection of transferred electrons eventually back to electronically depleted Au+ positive ions. Bridged by adsorbed molecules, electrons were repeatedly circulated back and forth in plasmonic nanoreactors, where the collected light was eventually converted into chemical energy.

Metabolomic profiling and assessment of antimicrobial, antioxidant and genotoxic potential of Unonopsis guatterioides R.E.Fr. (Annonaceae) fruits

Unonopsis guatterioides (A.DC.) R.E.Fr., is found mainly in the Pantanal, Cerrado and Amazon biomes and, some species of this genus are used in folk medicine. The analysis by HPLC-ESI-MS revealed the presence of alkaloids previously reported for Unonopsis genus, such as asimilobine, anonaine, nornuciferine, glaucine and norglaucine. In contrast, the heliamine, norjuziphine and anomuricine alkaloids are being reported for the first time in the Unonopsis genus, while this is the first report of azafluoranthene alkaloid triclisine in the Annonaceae family. These results showed the promising application of mass spectrometric monitoring of complex extracts in the search for novel natural products for the food, pharmaceutical, and cosmetic industries, thus simplifying phytochemical analysis. Bioactive analysis based on antioxidant activity indicated that ethanolic extracts of the peels and pulps of the fruits from U. guatterioides showed low scavenging activity against the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), with IC50 values > 100 μg*mL−1, using as reference the ascorbic acid (IC50 < 50 μg*mL−1). These results are in concordance with the chemical profiles, whose major compounds proved to be O-substituted phenolic derivatives. Furthermore, ethanolic extracts of the peels (UGP-1) and pulps (UGP-2) of the fruits from U. guatterioides showed weak activity against Staphylococcus pseudintermedius, S. aureus and S. epidermidis, with MIC values above 1000 μg*mL−1. The combination of the ethanolic extract of the pulps of fruits from U. guatterioides and ampicillin resulted in an additive effect (FICI = 1.0), when tested against S. aureus and a strain of S. epidermidis. These results suggest that the pulps' ethanolic extract, when combined with the antibiotic ampicillin, can strengthen the therapy for S. aureus and S. epidermidis infection. Additionally, no genotoxic activity of the ethanolic extracts of fruits from U. guatterioides (UGP-1 and UGP-2) was detected at the tested concentrations (0.25, 1.25, 2.5 and 5.0 mg*mL−1). The genotoxic property was performed using the Somatic Mutation and Recombination assay (SMART Test), in vivo, in somatic cells of Drosophila melanogaster.

Rate constant of H atom reaction with Br2 determined between 220 and 950 K

Rate constant of the reaction H + Br2 → HBr + Br (1) has been measured in a discharge flow reactor with mass spectrometric monitoring of the species involved: k1 = 7.06 × 10−11 (T/298)0.88 exp(182/T) cm3 molecule−1 s−1 at T = 220 – 950 K (with estimated total uncertainty on the rate constant of 15% at all temperatures). The kinetic data in the present work challenge previous experimental measurements and support the most recent theoretical calculations.

Technologies for Direct Detection of Covalent Protein-Drug Adducts.

In the past two decades, drug candidates with a covalent binding mode have gained the interest of medicinal chemists, as several covalent anticancer drugs have successfully reached the clinic. As a covalent binding mode changes the relevant parameters to rank inhibitor potency and investigate structure-activity relationship (SAR), it is important to gather experimental evidence on the existence of a covalent protein-drug adduct. In this work, we review established methods and technologies for the direct detection of a covalent protein-drug adduct, illustrated with examples from (recent) drug development endeavors. These technologies include subjecting covalent drug candidates to mass spectrometric (MS) analysis, protein crystallography, or monitoring intrinsic spectroscopic properties of the ligand upon covalent adduct formation. Alternatively, chemical modification of the covalent ligand is required to detect covalent adducts by NMR analysis or activity-based protein profiling (ABPP). Some techniques are more informative than others and can also elucidate the modified amino acid residue or bond layout. We will discuss the compatibility of these techniques with reversible covalent binding modes and the possibilities to evaluate reversibility or obtain kinetic parameters. Finally, we expand upon current challenges and future applications. Overall, these analytical techniques present an integral part of covalent drug development in this exciting new era of drug discovery.

SCRN1: A cerebrospinal fluid biomarker correlating with tau in Alzheimer's disease.

Secernin-1 (SCRN1) is a neuronal protein that co-localizes with neurofibrillary tangles in Alzheimer's disease (AD), but not with tau inclusions in corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), or Pick's disease. We measured SCRN1 concentration in cerebrospinal fluid (CSF) using a novel mass spectrometric parallel reaction monitoring method in three clinical cohorts comprising patients with neurochemically characterized AD (n=25) and controls (n=28), clinically diagnosed Parkinson's disease (PD; n=38), multiple system atrophy (MSA; n=31), PSP (n=20), CBD (n=8), healthy controls (n=37), and neuropathology-confirmed AD (n=47). CSF SCRN1 was significantly increased in AD (P<0.01, fold change=1.4) compared to controls (receiver operating characteristic area under the curve=0.78) but not in CBD, PSP, PD, or MSA. CSF SCRN1 positively correlated with CSF total tau (R=0.78, P=1.1×10-13 ), phosphorylated tau181 (R=0.64, P=3.2×10-8 ), and Braak stage and negatively correlated with Mini-Mental State Examination score. CSF SCRN1 is a candidate biomarker of AD, reflecting tau pathology. We developed a parallel reaction monitoring assay to measure secernin-1 (SCRN1) in cerebrospinal fluid (CSF). CSF SCRN1 was increased in Alzheimer's disease compared to healthy controls. CSF SCRN1 remained unchanged in Parkinson's disease, multiple system atrophy, progressive supranuclear palsy, or corticobasal degeneration compared to controls. CSF SCRN1 correlated strongly with CSF phosphorylated tau and total tau. CSF SCRN1 increased across Braak stages and negatively correlated with Mini-Mental State Examination score.

Sphingolipidomics of Bovine Pink Eye: A Pilot Study.

Simple SummaryThe liquid tear film, which protects the eye from the environment, is a dynamic fluid containing a large number of complex lipids. Disruptions of these lipids by infections can result in damage to the eye and ultimately blindness. In this study we characterized various lipid subfamilies present in the tear film of the eye and the effect of pink eye infections in cattle. Our findings demonstrate that the pink eye infections dramatically decrease the levels of lipids in the tear film covering the eye and suggest that this is a major factor in the development of blindness in infected cattle.Sphingolipids are essential structural components of tear film that protect the surface of the eye from dehydration. A detailed analysis of the effects of pink eye infections on the sphingolipidome in cattle has not previously been undertaken. We recently published a new assay utilizing high-resolution mass spectrometric monitoring of the chloride adducts of sphingolipids that provides enhanced sensitivity and specificity. Utilizing this assay, we monitored decreases in the levels of tear film ceramides with short-chain fatty acids, hydroxy-ceramides, phytoceramides, and hydroxy-phytoceramides. Dihydroceramide levels were unaltered and increased levels of ceramides with long-chain fatty acids (24:0 and 24:1) were monitored in cattle with pink eye. The data from this pilot study (n = 8 controls and 8 pink eye) demonstrate a major disruption of the lipid tear film layer in pink eye disease, that can result in severe eye irritation and damage.

Clinical Assay for the Early Detection of Colorectal Cancer Using Mass Spectrometric Wheat Germ Agglutinin Multiple Reaction Monitoring.

Simple SummaryColorectal cancer (CRC) is currently the third leading cause of cancer death worldwide. Early diagnosis of CRC is important for increasing the opportunity for treatment and receiving a good prognosis. The aim of our study was to develop a detection method that combined wheat germ agglutinin (WGA) chromatography with mass spectrometry (MS) for early detection of CRC. Further, machine learning algorithms and logistic regression were applied to combine multiple biomarkers we discovered. We validated in a population of 286 plasma samples the diagnostic performance of peptides corresponding to WGA-captured protein and its combination, which received a sensitivity of 84.5% and a specificity of 97.5% in the diagnoses of CRC. Proteomic biomarkers combined with algorithms can provide a powerful tool for discriminating patients with CRC and health controls (HCs). Measurements of WGA-captured PF4, ITIH4, and APOE with MS are then useful for early detection of CRC. Additionally, our study revealed the potential of applying lectin chromatography with MS for disease diagnosis.Colorectal cancer (CRC) is currently the third leading cause of cancer-related mortality in the world. U.S. Food and Drug Administration-approved circulating tumor markers, including carcinoembryonic antigen, carbohydrate antigen (CA) 19-9 and CA125 were used as prognostic biomarkers of CRC that attributed to low sensitivity in diagnosis of CRC. Therefore, our purpose is to develop a novel strategy for novel clinical biomarkers for early CRC diagnosis. We used mass spectrometry (MS) methods such as nanoLC-MS/MS, targeted LC-MS/MS, and stable isotope-labeled multiple reaction monitoring (MRM) MS coupled to test machine learning algorithms and logistic regression to analyze plasma samples from patients with early-stage CRC, late-stage CRC, and healthy controls (HCs). On the basis of our methods, 356 peptides were identified, 6 differential expressed peptides were verified, and finally three peptides corresponding wheat germ agglutinin (WGA)-captured proteins were semi-quantitated in 286 plasma samples (80 HCs and 206 CRCs). The novel peptide biomarkers combination of PF454–62, ITIH4429–438, and APOE198–207 achieved sensitivity 84.5%, specificity 97.5% and an AUC of 0.96 in CRC diagnosis. In conclusion, our study demonstrated that WGA-captured plasma PF454–62, ITIH4429–438, and APOE198–207 levels in combination may serve as highly effective early diagnostic biomarkers for patients with CRC.

Emergence of Delta-8 Tetrahydrocannabinol in DUID Investigation Casework: Method Development, Validation and Application.

Cannabinoid is the most frequently reported illicit drug class in Driving Under the Influence of Drugs (DUID) investigation casework. In recent years, our laboratory observed an increasing rate of overlapping peaks for the cannabinoid confirmation performed using 2D high-performance liquid chromatography (LC)--tandem mass spectrometry (MS-MS). Starting in early 2018, the incidence of unresolved interfering substances increased, contributing to a higher rate of canceled testing that peaked at 3.7% in February 2019. The observed interference demonstrates a distinctive pattern affecting the identification and quantification of both delta-9 tetrahydrocannabinol (THC) and delta-9 carboxy THC. An improved quantitative method was developed and validated to separate delta-8 and -9 isomers and their metabolites in blood. All acceptance criteria were met, with identical measurement ranges from the original method (lower limit of quantitation: 0.5 ng/mL for delta-9 THC, 1.0 ng/mL for 11-hydroxy delta-9 THC and 5.0 ng/mL for delta-9 carboxy THC). Cannabinoids were extracted from whole blood using liquid-liquid extraction, separated in a 2D LC system over a run time of 10 min and detected by an MS-MS system equipped with ESI source operating in positive ionization mode with scheduled multiple reaction mass spectrometric monitoring. The LC system consisted of a pair of Phenomenex® SecurityGuard™ C6 Phenyl (4 × 2 mm) cartridges for extracting the compounds with 5 mM ammonium formate buffer in deionized (DI) water and 0.1% formic acid in methanol as mobile phase, and a Phenomenex® Kinetex C18 column (100 × 3 mm) with 0.1% formic acid in DI water and 0.1% formic acid in methanol for LC separation at 45°C. Each set of isomers was fully resolved by the longer run-time method. To the authors' knowledge, this is the first report of a method that successfully quantitates these primary cannabinoids in blood specimens where significant concentrations of both delta-9 and delta-8 isomers are present.

Mass Spectrometric Monitoring of Plant Hormone Cross Talk During Biotic Stress Responses in Potato (Solanum tuberosum L.).

The potato is among the most important food crops in the world and of incalculable value for global food security. In 2012, the crop area for potato in Northern and Western Europe reached almost 1 million ha and a production of over 37 million tons with an average yield between 18 and 45tons/ha. However, current potato production is put in jeopardy by a number of important biotic stress factors including late blight (Phytophthora infestans), which was responsible for the disastrous Irish potato famine during 1843-1845. P. infestans shows a remarkable capacity for adaptation with respect to host genotype and applied fungicides. This has made disease management to become more and more difficult and put substantial emphasis on gaining more detailed insight into the molecular bases of plant pathogen interactions, in order to find more sophisticated ways for biological pest control. The plant hormones jasmonic acid (JA) and salicylic acid (SA) play central roles in the regulation of plant responses to biotic foes. In addition, other phytohormones including auxins and abscisic acid (ABA) have also been associated with plant defense responses. For this reason, the parallel analysis of multiple plant hormones in small tissue amounts represents an important field of research in contemporary plant sciences. Here, we describe a highly sensitive and accurate method for the quantitative analysis of ABA, JA, SA, and indole-3-acetic acid in potato plants by gas chromatography-coupled tandem mass spectrometry (GC-MS/MS).

Comprehensive thermochemical study of sublimation, melting and vaporization of scandium(III) beta-diketonates

The pressure of saturated and unsaturated vapor over solid and liquid scandium(III) complexes with acetylacetone Sc(acac)3, trifluoroacetylacetone Sc(tfac)3, and hexafluoroacetylacetone Sc(hfac)3 has been measured using a static method with glass membrane-gauge manometers and Knudsen effusion method with mass spectrometric gas phase monitoring. The temperatures and enthalpies of melting were measured for these compounds by differential scanning calorimetry. As a result of this study the thermal stabilities of investigated complexes were established, the temperature dependences of saturated vapor pressure were obtained, the thermodynamic characteristics of processes under study (ΔprH°T, ΔprS°T) were calculated. Thermal properties of these compounds were compared with literature data.

Facile in-situ polymerization of polyaniline-functionalized melamine sponge preparation for mass spectrometric monitoring of perfluorooctanoic acid and perfluorooctane sulfonate from biological samples

In this present work, a novel polyaniline-functionalized melamine sponge (PMs) was successfully prepared using a simple unstirred in-situ polymerization process. The PMs was characterized using a scanning electron microscope and contact angle measurements. Its adsorption performance was initially determined via dye adsorption assays, and the conditions affecting the synthesis including polymerization time, acidity, molar ratio, and number and sizes of raw melamine sponge were optimized. The PMs was then used as an efficient adsorbent for the development of a novel, low-cost method for the detection of two representative perfluorinated chemicals, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), using ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QqQ-MS/MS) with the internal standard method. To achieve the best extraction efficiency with this method, several variables were optimized, including adsorption time, pH value, the number of PMs, and desorption conditions. Calibration graphs showed a good linear degree at concentration ranging from 0.1 to 50 μg L−1 for PFOA and 0.01–10 μg L−1 for PFOS, with a coefficient of detection R2 = 0.998. The intra-day and inter-day relative standard deviations were found to range from 5.9% to 8.2% for PFOA, and 5.5% to 7.7% for PFOS. Under these optimized conditions, the method was successfully used to measure PFOA and PFOS content in real human serum and urine samples, with average spiked recoveries ranging from 79% and 91% for PFOA, and 81% to 87% for PFOS.

Difference in Involved and Uninvolved Free Light Chain (dFLC) of Less Than 1mg/DL Early Post Risk Adapted Melphalan and Autologous Stem Cell Transplantation (RA-ASCT) Predicts Renal Response at 1 Year in Light Chain (AL) Amyloidosis

Background: Deep and durable hematologic remissions following RA-ASCT are associated with improved organ function and extended overall survival (OS) in AL amyloidosis. Achieving at least a very good partial response (VGPR) defined by a dFLC &lt;4mg/dL is an accepted goal of therapy based on favorable outcomes, including improved renal survival (REF: Palladini JCO 2012, Palladini Blood 2014). Recently more profound clonal suppression as indicated by no evidence of minimal residual plasma cell disease (MRD) in bone marrow (BM) (Muchtar Blood 2017) and achieving dFLC &lt;1mg/dL (Manwani Blood 2018) have shown additional benefit. While depth of hematologic response by standard criteria are important, this study assessed additional factors that influence renal response and time to renal response. Methods: All patients (pts) with AL and renal involvement (biopsy proven renal tissue diagnosis and/or 24hr proteinuria &gt;500mg/day) undergoing RA-ASCT at Memorial Sloan Kettering Cancer Center between January 1, 2007 to December 31, 2016 were included. Pts with follow up less than 12 months post RA-ASCT, hemodialysis prior to RA-ASCT and Waldenstrom macroglobulinemia were excluded. Melphalan dose was assigned based on age, cardiac involvement and renal compromise (Landau Leukemia 2013). Hematologic response was assessed at 3 and 12 months (mos) post RA-ASCT (Palladini JCO 2012) and those with less than complete response (CR) were offered consolidation therapy with bortezomib and dexamethasone (BD). All pts underwent serial organ function assessment (Palladini Blood 2014). Logistic regression models were used to assess association with renal response by 12 mos. Covariates for adjustment in multivariate models were chosen based on univariate analyses and clinical relevance. Results: Sixty-four patients with renal AL meeting the inclusion criteria were identified; 3 pts died within a year post RA-ASCT were excluded. Median age (range) was 61 years (44-73), M:F 49%:51%, white 90% and 34% had cardiac involvement. Median (IQR) 24 hr proteinuria pre RA-ASCT was 5014 mg/day (2632-7514) and eGFR 68 ml/min/1.73 m2 (44-91). Renal amyloid stage I:II:III was 33%:52%:15%. Mayo cardiac stage (2004) I:II:III was 28%:61%:11% and revised Mayo stage (2012) I:II:III:IV was 13%:57%:21%:8%. Median BM plasma cells pre RA-ASCT was 9% (IQR 2-14%). 46% pts received treatment prior to ASCT. Melphalan dose (mg/m2) 200:140:100 was 44%:43%:11%. 46% pts received BD consolidation. Hematologic response at 3 mos post RA-ASCT was CR 44%, VGPR 29%, partial response (PR) 20% and stable disease (SD) 7%. MRD in BM by 10-color flow cytometry was assessed in 33 pts and 13 (39%) were MRD negative. dFLC &lt;1mg/dL was achieved in 63% of pts. Renal response by 12 mos following RA-ASCT was achieved in 32 pts (53%). Median (IQR) time to renal response in these pts was 5.8 mos (5.1 - 11.3). Amongst renal responders, 50% were in CR, 53% had MRD negative BM (of 15 pts) and 78% with dFLC &lt;1mg/dL early post RA-ASCT. In pts who achieved dFLC &lt;1mg/dL early post RA-ASCT, 66% had renal response. By univariate analysis (Table 1) OR (95% CI) Mayo cardiac Stage (2004) II and III 0.23 (0.07-0.83, p=0.025), early post RA-ASCT dFLC &lt;1mg/dL 3.00 ( 1.01-8.93, p=0.048), VGPR early post RA-ASCT 7.80 (1.69-36.06, p=0.009), dFLC &lt;1mg/dL at 12 mos 7.20 (2.14-24.21, p=0.001) and CR at 12 mos 10.27 (1.14-92.26, p=0.038) were significantly associated with renal response. Neither renal stage, Mayo stage (2012), MRD negativity, melphalan dose nor consolidation was associated with renal response. By multivariate analysis (Table 2), early post RA-ASCT dFLC &lt;1mg/dL continued to be the most significant factor predicting renal response, OR (95% CI) 4.52 (1.26-16.24, p=0.021), when adjusted for renal amyloid stage and Mayo cardiac stage (2004). Conclusion: In this single center study, we report that RA-ASCT results in renal response in more than half (53%) of the patients at 1 year. Achieving dFLC &lt;1mg/dL early post ASCT is significantly associated with renal response. Renal response is independent of baseline proteinuria and BM plasma cells or MRD status post ASCT. Our study supports that pathologic entity in organ damage is not the plasma cells but rather light chains. Further studies using dFLC &lt;1mg/dL should be evaluated in organ response. Mass spectrometric light chain monitoring may even be more sensitive and could potentially serve as a non-invasive way to measure disease burden. Disclosures Shah: Janssen: Research Funding; Amgen: Research Funding. Hassoun:Janssen: Research Funding; Celgene: Research Funding; Novartis: Consultancy. Giralt:Celgene: Consultancy, Research Funding; Takeda: Consultancy; Sanofi: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. Landau:Pfizer: Membership on an entity's Board of Directors or advisory committees; Prothena: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Glycan Markers of Human Stem Cells Assigned with Beam Search Arrays*[S

Glycan antigens recognized by monoclonal antibodies have served as stem cell markers. To understand regulation of their biosynthesis and their roles in stem cell behavior precise assignments are required. We have applied state-of-the-art glycan array technologies to compare the glycans bound by five antibodies that recognize carbohydrates on human stem cells. These are: FC10.2, TRA-1-60, TRA-1-81, anti-i and R-10G. Microarray analyses with a panel of sequence-defined glycans corroborate that FC10.2, TRA-1-60, TRA-1-81 recognize the type 1-(Galβ-3GlcNAc)-terminating backbone sequence, Galβ-3GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and anti-i, the type 2-(Galβ-4GlcNAc) analog, Galβ-4GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and we determine substituents they can accommodate. They differ from R-10G, which requires sulfate. By Beam Search approach, starting with an antigen-positive keratan sulfate polysaccharide, followed by targeted iterative microarray analyses of glycan populations released with keratanases and mass spectrometric monitoring, R-10G is assigned as a mono-sulfated type 2 chain with 6-sulfation at the penultimate N-acetylglucosamine, Galβ-4GlcNAc(6S)β-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc. Microarray analyses using newly synthesized glycans corroborate the assignment of this unique determinant raising questions regarding involvement as a ligand in the stem cell niche.

Simple 3D printed stainless steel microreactors for online mass spectrometric analysis

A simple flow chemistry microreactor with an electrospray ionization tip for real time mass spectrometric reaction monitoring is introduced. The microreactor was fabricated by a laser-based additive manufacturing technique from acid-resistant stainless steel 316L. The functionality of the microreactor was investigated by using an inverse electron demand Diels-Alder and subsequent retro Diels-Alder reaction for testing. Challenges and problems encountered are discussed and improvements proposed. Adsorption of reagents to the rough stainless steel channel walls, short length of the reaction channel, and making a proper ESI tip present challenges, but the microreactor is potentially useful as a disposable device.

Confounding contaminants in mass spectrometric reaction monitoring

Reactions carried out in the presence of rubber septa run the risk of additives being leached out by the solvent. Normally, such species are present at low enough levels that they do not interfere with the reaction significantly. However, when studying reactions using sensitive methods such as mass spectrometry, the appearance of even trace amounts of material can confuse dynamic analyses of reactions. A wide variety of additives are present in rubber along with the polymer: antioxidants, dyes, detergent, and vulcanization agents, and these are all especially problematic in negative ion mode. A redesigned Schlenk flask for pressurized sample infusion (PSI) is presented as a means of practically eliminating the presence of contaminants during reaction analyses.

Identification of Multisialylated LacdiNAc Structures as Highly Prostate Cancer Specific Glycan Signatures on PSA.

Serum prostate-specific antigen (PSA) test is the current gold standard for screening and diagnosis of prostate cancer (PCa), while overdiagnosis and overtreatment are social problems. In order to improve the specificity and exclude a false positive diagnosis in PSA test, PCa-specific glycosylation subtypes of PSA were explored using in-depth quantitative profiling of PSA glycoforms based on mass spectrometric oxonium ion monitoring technology. As a result of analysis using sera from 15 PCa or 15 benign prostate hyperplasia (BPH) patients whose PSA levels were in the "gray zone" (4.0-10.0 ng/mL), 52 glycan structures on PSA were quantitatively observed. We found that abundance of multisialylated LacdiNAc (GalNAcβ1-4GlcNAc) structures were significantly upregulated in the PCa group compared to the BPH group. A couple of those glycoforms were then extracted and subjected to establish a novel PCa-specific diagnosis model (PSA G-index). When the diagnostic power was assessed using an independent validation sample set (15 PCa and 15 BPH patients in the PSA gray zone), an AUC of PSA G-index was 1.00, while that of total PSA or PSA f/T ratio was 0.50 or 0.60, respectively. Moreover, both PSA glycoforms showed significant correlation with Gleason scores. Lectin histochemical staining analysis also showed that PCa cells overexpressed glycoproteins containing LacdiNAc and sialic acids moieties. Thus, PSA G-index could serve as not only an effective secondary screening method to exclude false positive diagnosis in PSA screening, but also a potential grading biomarker for PCa.

Extending PTR based breath analysis to real-time monitoring of reactive volatile organic compounds.

Reactive exhaled volatile organic compounds (VOCs) such as nitrogen- and sulfur-containing substances may be related to diseases, metabolic processes and bacterial activity. As these compounds may interact with any surface of the analytical system, time-resolved monitoring and reliable quantification is difficult. We describe a proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) based analytical method for direct breath-resolved monitoring of reactive compounds. Aliphatic amines were used as test substances. Matrix adapted gas standards were generated by means of a liquid calibration unit. Calibration conditions were adapted in terms of materials, temperature and equilibration time. PTR-ToF-MS conditions were optimized in terms of inlet materials, transfer line and drift tube temperature and drift tube reduced electric field (E/N). Optimized PTR conditions in combination with inert materials and high temperatures considerably reduced the interactions of compounds with the surfaces of the analytical system. Good linearity (R2 > 0.99, RSDs < 5%) with LODs between 0.15 ppbV and 1.23 ppbV and LOQs between 0.24 ppbV and 1.94 ppbV could be achieved. The method was then applied to breath-resolved monitoring of reactive compounds in 17 healthy subjects after high and low oral protein challenge. Exhaled concentrations of trimethylamine, indole, methanethiol, dimethylsulfide, acetone, 2-propanol, 2-butanone and phenol showed significant changes after protein intake. Methanethiol concentrations increased 6-fold within minutes after the protein intake. Optimization of methods and instrument design enabled reliable breath-resolved PTR-MS based analysis of exhaled reactive VOCs in the sub-ppbV range. Continuous in vivo monitoring of exhaled amines and sulphur containing compounds may provide novel non-invasive insight into endogenous and gut bacteria driven protein metabolism.

In Situ Mass Spectrometric Reaction Monitoring of Atmospheric Corrosion Processes

A set‐up for in situ mass spectrometric reaction monitoring of gas phase mediated reactions is presented. Important constructive parameters for a reaction chamber for the investigation of atmospheric corrosion processes is discussed. The presented cell is especially well suited for atmospheric corrosion studies but it can easily be modified to be applicable in other fields of research as well. The atmospheric corrosion of electrolytically deposited zinc surfaces on steel during wet‐ and dry‐cycles is investigated as a model reaction. Corrosion rates as well as current densities during the first wet‐ and dry cycle are calculated and discussed. Complementary scanning Kelvin probe (SKP) measurements are performed to show the electrochemical changes occurring at the metal/air‐interface throughout the corrosion process. Contact potential difference versus distance measurements performed in advance to determine the maximum measurement distance, which allows investigating rough salt loaded sample surfaces via SKP surface scans.