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The specific positions of carbon–carbon double bond(s) within an unsaturated fatty acid exert a significant effect on the physical and chemical properties of the lipid that ultimately inform its biological function(s). Contemporary liquid chromatography–mass spectrometry (MS) strategies based on electrospray ionization coupled to tandem MS can easily detect fatty acyl lipids but generally cannot reveal those specific site(s) of unsaturation. Herein, we describe a novel and versatile workflow whereby fatty acids are first converted to fixed charge N-(4-aminomethylphenyl)pyridinium (AMPP) derivatives and subsequently subjected to ozone-induced dissociation (OzID) on a modified triple quadrupole mass spectrometer. The AMPP modification enhances the detection of fatty acids introduced by direct infusion. Fragmentation of the derivatized fatty acids also provides diagnostic fragment ions upon collision-induced dissociation that can be targeted in precursor ion scans to subsequently trigger OzID analyses in an automated data-dependent workflow. It is these OzID analyses that provide unambiguous assignment of carbon–carbon double bond locations in the AMPP-derivatized fatty acids. The performance of this analysis pipeline is assessed in profiling the patterns of unsaturation in fatty acids within the complex biological secretion vernix caseosa. This analysis uncovers significant isomeric diversity within the fatty acid pool of this sample, including a number of hitherto unreported double bond positional isomers that hint at the activity of potentially new metabolic pathways. [ABSTRACT FROM AUTHOR]

Peptides represent a promising modality for the design of novel therapeutics that can potentially modulate traditionally non-druggable targets. Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are two large families that are being explored extensively as drug delivery vehicles, imaging reagents, or therapeutic treatments for various diseases. Many CPPs and AMPs are cationic among which a significant portion is extremely basic and hydrophilic (e.g., nona-arginine). Despite their attractive therapeutic potential, it remains challenging to directly analyze and quantify these super cationic peptides from biological matrices due to their poor chromatographic behavior and MS response. Herein, we describe a generic method that combines solid phase extraction and LC-MS/MS for analysis of these peptides. As demonstrated, using a dozen strongly basic peptides, low μM concentration of perfluoropentanoic acid (PFPeA) in the mobile phase enabled excellent compound chromatographic retention, thus avoiding co-elution with solvent front ion suppressants. PFPeA also had a charge reduction effect that allowed the selection of parent/ion fragment pairs in the higher m/z region to further reduce potential low molecular weight interferences. When the method was coupled to the optimized sample extraction process, we routinely achieved low digit ng/ml sensitivity for peptides in plasma/tissue. The method allowed an efficient evaluation of plasma stability of CPPs/AMPs without fluorescence derivatization or other tagging methods. Importantly, using the widely studied HIV-TAT CPP as an example, the method enabled us to directly assess its pharmacokinetics and tissue distribution in preclinical animal models. [ABSTRACT FROM AUTHOR]

N-linked glycosylation is one of the most common protein PTMs, and the topological structure (monosaccharide composition and sequence as well as glycosidic linkages) of N-glycans is vital information to understand their biological functions and roles. Tandem mass spectrometry has been widely used for topological structure characterization of N-glycans, where comprehensive understanding of fragmentation pathways and characteristics of product ions are essential to achieve best interpretation of MS/MS data and highest confidence of identification. Here, we report our glycomic study of N-glycome of mouse brain as well as fragmentation pathway analysis of the identified N-glycans. With LC-MS/MS analysis at both the positive and negative ESI modes together with our recently developed N-glycan database search engine GlySeeker, 221 unique N-glycans with putative topological structures were identified with target-decoy searches and number of best hits of 1. Analysis of fragmentation pathways and characteristics of product ions of permethylated N-glycans in the positive mode and native N-glycans in the negative mode were further carried out. The reported N-glycans serve as a basic reference for future glycosylation study of mouse brain; and in general database search of tandem mass spectra of N-glycans, B/Y/Z ions should be preferentially considered for the permethylated form in the positive mode and B/C/Z ions for the native form in the negative mode. [ABSTRACT FROM AUTHOR]

Multiple ion transition summation of isotopologues (MITSI) is an adaptable and easy-to-implement methodology for improving analytical sensitivity, especially for halogenated compounds and otherwise abundant isotopologues. This novel application of signal summing was applied to measure and quantitate the two most abundant ion transitions of two isotopologues of N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (1DCV), a urinary metabolite of trichloroethylene (TCE). Because 1DCV is dichlorinated, only approximately half of the total potential signal is quantifiable when the monoisotopic ion transition (i.e., m/z 256 → 127 for 35Cl2) is monitored. By summing the intensity of a separate and high-abundance 1DCV isotopologue ion transition (i.e., m/z 258 → 129 to include 35Cl and 37Cl), overall signal intensity increased by over 70%. This summation technique improved the analytical sensitivity and limit of detection (LOD) by factors of 2.3 and 2.9, respectively, compared to monitoring the two transitions separately, without summation. Separation and detection were performed using liquid chromatography–tandem mass spectrometry (LC-MS/MS) in negative-ion mode with scheduled selected reaction monitoring. This approach was verified for accuracy and precision using two quality control materials. In addition, we derived a modified signal summation equation to calculate predicted signal enhancements specific to the MITSI approach. [ABSTRACT FROM AUTHOR]

We report a reverse phase chromatography mass spectrometry (LC-MS) method for simultaneous quantification of nucleosides and nucleotides from biological samples, where compound identification was achieved by a tier-wise approach and compound quantification was achieved via external calibration. A total of 65 authentic standards of nucleosides and nucleotides were used for the platform development. The limit of detection (LOD) of those compounds ranged from 0.05 nmol/L to 1.25 μmol/L, and their limit of quantification (LOQ) ranged from 0.10 nmol/L to 2.50 μmol/L. Using the developed method, nucleosides and nucleotides from human plasma, human urine, and rat liver were quantified. Seventy-nine nucleosides and nucleotides were identified from human urine and 28 of them were quantified with concentrations of 13.0 nmol/L–151 μmol/L. Fifty-five nucleosides and nucleotides were identified from human plasma and 22 of them were quantified with concentrations of 1.21 nmol/L–8.54 μmol/L. Fifty-one nucleosides and nucleotides were identified from rat liver and 23 were quantified with concentrations of 1.03 nmol/L–31.7 μmol/L. These results demonstrate that the developed method can be used to investigate the concentration change of nucleosides and nucleotides in biological samples for the purposes of biomarker discovery or elucidation of disease mechanisms. [ABSTRACT FROM AUTHOR]

Non-targeted screening (e.g., suspected-target) is emerging as an attractive tool to investigate the occurrence of contaminants in food. The sample preparation and instrument analysis steps are known to influence the identification of analytes with non-targeted workflows, especially for complex matrices. However, for methods based on mass spectrometry, the impact of the post-analysis data treatment (e.g., feature extraction) on the capacity to correctly identify a contaminant at trace level is currently not well understood. The aim of the study was to investigate the influence of seven post-analysis data treatment parameters on the non-targeted identification of trace contaminants in honey using high-performance liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). Seven compounds reported as veterinary drugs for honeybees were applied as model compounds. Among the parameters studied, the expansion window for chromatogram extraction and the average scans included in the spectra influenced significantly the identification process results. The optimized data treatment was applied to the non-targeted screening of veterinary drugs, pesticides, and other contaminants in 55 honey samples as a proof of concept. Among the 43 compounds included in a library of honey-related compounds that was used for screening, eight compounds were tentatively identified in at least one honey sample. The tentative identity of two of these compounds (tylosin A and hydroxymethylfurfural) was further confirmed with analytical standards. [ABSTRACT FROM AUTHOR]

Here, we report a continuous flow-based ionization method, capillary vibrating sharp-edge spray ionization (cVSSI), that nebulizes liquid sample directly at the outlet of a capillary without using high-speed nebulization gas or a high electrical field. cVSSI is built upon the recently reported VSSI principle which nebulizes bulk liquid using vibrating sharp-edges. By attaching a short piece of fused silica capillary on top of the vibrating glass slide in VSSI, liquid is nebulized at the outlet of the capillary as the result of the vibration. Utilizing standard 360-μm OD/100-μm ID capillary, cVSSI works with a wide range of flow rates from 1 μL/min to 1 mL/min. The power consumption is as low as 130 mW. ESI-like MS spectra are obtained for small molecules, peptides, and proteins. Five orders of magnitude linear response for acetaminophen solution is achieved with a limit of detection (LOD) of 3 nM. cVSSI is also demonstrated to be compatible with LC-MS analysis. Two LC-MS applications are demonstrated with cVSSI: (1) separation and detection of a mixture of small molecules and (2) bottom-up proteomics using a protein digest. A mixture of nine common metabolites was appropriately separated and detected using LC-cVSSI-MS. In the bottom-up experiment, 78 peptides were detected using LC-cVSSI-MS/MS with a protein coverage of 100% for cytochrome c, which is comparable with the coverage obtained using LC-ESI-MS. cVSSI offers a means of interfacing LC or other continuous flow-based applications to mass spectrometers with the salient features of voltage-free, flexibility, small footprint, and low power consumption. [ABSTRACT FROM AUTHOR]

Nanoflow liquid chromatography coupled with a captive spray ionization time-of-flight tandem mass spectrometer (nLC/CSI-QTOF-MS/MS) was used in the structural determination of polar steroid compounds of starfish Lethasterias fusca. A total of 207 compounds including 106 asterosaponins, 81 glycosides of polyhydroxysteroids, and 14 polyhydroxylated steroids were detected and characterized by MS and MS/MS. Twenty compounds among them were unambiguously identified using authentic standard compounds, isolated earlier from this and other starfish species. The other compounds were tentatively characterized by accurate mass measurement and comparing retention times and characteristic MS/MS fragmentation patterns with reference standards. Moreover, fragmentation behaviors of a series of pure standards of starfish polar steroids and polyhydroxysteroid compounds detected in L. fusca have been extensively investigated and characteristic fragmentation pathways were described and used for the characterization of unknown compounds. [ABSTRACT FROM AUTHOR]

Mass spectrometry-based quantification method has advanced rapidly. In general, the methods for accurate quantification rely on the use of authentic target compounds or isotope-labeled compounds as standards, which might be not available or difficult to synthesize. To tackle this grand challenge, this paper presents a novel approach, based on electrochemistry (EC) combined with mass spectrometry (MS). In this approach, a target compound is allowed to undergo electrochemical oxidation and then subject to MS analysis. The oxidation current recorded from electrochemistry (EC) measurement provides information about the amount of the oxidized analyte, based on the Faraday's Law. On the other hand, the oxidation reaction yield can be determined from the analyte MS signal changes upon electrolysis. Therefore, the total amount of analyte can be determined. In combination with liquid chromatography (LC), the method can be applicable to mixture analysis. The striking strength of such a method for quantitation is that neither standard compound nor calibration curve is required. Various analyte molecules such as dopamine, norepinephrine, and rutin as well as peptide glutathione in low quantity were successfully quantified using our method with the quantification error ranging from − 2.6 to + 4.6%. Analyte in a complicated matrix (e.g., uric acid in urine) was also accurately measured. Graphical Abstract [ABSTRACT FROM AUTHOR]

D-Amino acids are important biological molecules. Improved analytical methods for their resolution and quantification remain of keen interest. In this study, we investigated the use of Marfey's reagent (chiral) derivatization coupled with LC-MS/MS-based separation and detection of the resulting diastereomers for quantification of the 19 common L- and D-amino acids and glycine. Standard formic acid (pH 2)-based separations on reverse phase media were unable to separate all 19 amino acid DL pairs. In contrast, a water/acetonitrile/ammonium acetate (pH 6.5) solvent system allowed all 19 amino acid DL pairs to be chromatographically resolved on a 30 min gradient, with negative mode detection at pH 6.5 giving good sensitivity. Derivatization reaction rates between amino acids varied substantially, with overnight derivatization required for some amino acids. Chromatography at pH 6.5 combined with MS/MS quantification in negative mode demonstrated good linearity over a wide concentration range for all 20 amino acids. Matrix effects, assessed with an MRSA extract, were negligible. Marfey's derivatized analytes were stable for 24 h at room temperature. This method was demonstrated by determining the levels of these analytes in mid-log phase MRSA extracts. This approach provides for the chromatographic resolution and MS/MS-based quantification of all 20 common L- and D-amino acids in complex matrices.Graphical Abstract [ABSTRACT FROM AUTHOR]

When characterizing components from ginseng, we found a vast number of multicharged anions presented in the liquid chromatography-mass spectrometry (LC-MS) chromatograms. The source of these anions is unclear yet, while ginsenosides, the major components of ginseng, are the main suspected type of molecules because of their sugar moiety. Our investigation using 14 pure ginsenosides affirmed that the multicharged anions were formed by ginsenosides rather than other types of ingredients in ginseng. Various anions could be observed for each ginsenoside. These anions contain ions ([M-2H]2−, [M+Adduct]2−), as well as those formed by polymerization of at least two ginsenosides, such as [nM-2H]2−, [nM-H+Adduct]2−, and [nM-3H]3−. The presence of so different types of ions from a ginsenoside explains the reason for the large number of anions in the LC-MS analysis of ginseng. We further found that formation of [nM-2H]2− ions was influenced by the number of sugar chains: ginsenosides containing two sugar chains produced all [nM-2H]2− ion types, whereas ginsenosides containing one sugar chain did not produce [2M-2H]2−. Thus, [2M-2H]2− and [3M-2H]2− can be utilized to rapidly identify monodesmosidic and/or bidesmosidic ginsenosides as joint diagnostic anions. The position of the glycosyl radical might be the key factor affecting the formation of multicharged multimer ions from monodesmosidic ginsenosides. Consequently, three groups of ginsenoside isomers were differentiated by characteristic [nM-2H]2− anions. Using concentration-dependent characteristics and collision-induced dissociation (CID), we confirmed that [nM-2H]2− ions are non-covalently bound multimers whose aggregation has marked distinction between monodesmosidic and bidesmosidic ginsenosides, accounting for the differentiated formation of [nM-2H]2− between them.Graphical Abstract [ABSTRACT FROM AUTHOR]

Current literature shows a gap for methods which can identify yeast sub-species (strains or serovars) in samples where there are no viable cells remaining. Presented here is a technique for the analysis of yeast supernatant, including solid phase extraction, data-dependent acquisition liquid chromatography/mass spectrometry (LC-MS), and two chemometric methods to identify and classify yeast strains. Five strains of Saccharomyces cerevisiae were successfully identified in various stages of growth. In addition, peptide/protein identification was performed, without the need for additional data acquisition.ᅟ [ABSTRACT FROM AUTHOR]

The analytical identification of positional isomers (e.g., 3-, N4-, 5-methylcytidine) within the > 160 different post-transcriptional modifications found in RNA can be challenging. Conventional liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approaches rely on chromatographic separation for accurate identification because the collision-induced dissociation (CID) mass spectra of these isomers nearly exclusively yield identical nucleobase ions (BH2+) from the same molecular ion (MH+). Here, we have explored higher-energy collisional dissociation (HCD) as an alternative fragmentation technique to generate more informative product ions that can be used to differentiate positional isomers. LC-MS/MS of modified nucleosides characterized using HCD led to the creation of structure- and HCD energy-specific fragmentation patterns that generated unique fingerprints, which can be used to identify individual positional isomers even when they cannot be separated chromatographically. While particularly useful for identifying positional isomers, the fingerprinting capabilities enabled by HCD also offer the potential to generate HPLC-independent spectral libraries for the rapid analysis of modified ribonucleosides.ᅟ [ABSTRACT FROM AUTHOR]

High-throughput methods to assess radiation exposure are a priority due to concerns that include nuclear power accidents, the spread of nuclear weapon capability, and the risk of terrorist attacks. Metabolomics, the assessment of small molecules in an easily accessible sample, is the most recent method to be applied for the identification of biomarkers of the biological radiation response with a useful dose-response profile. Profiling for biomarker identification is frequently done using an LC-MS platform which has limited throughput due to the time-consuming nature of chromatography. We present here a chromatography-free simplified method for quantitative analysis of seven metabolites in urine with radiation dose-response using urine samples provided from the Pannkuk et al. (2015) study of long-term (7-day) radiation response in nonhuman primates (NHP). The stable isotope dilution (SID) analytical method consists of sample preparation by strong cation exchange-solid phase extraction (SCX-SPE) to remove interferences and concentrate the metabolites of interest, followed by differential mobility spectrometry (DMS) ion filtration to select the ion of interest and reduce chemical background, followed by mass spectrometry (overall SID-SPE-DMS-MS). Since no chromatography is used, calibration curves were prepared rapidly, in under 2 h (including SPE) for six simultaneously analyzed radiation biomarkers. The seventh, creatinine, was measured separately after 2500× dilution. Creatinine plays a dual role, measuring kidney glomerular filtration rate (GFR), and indicating kidney damage at high doses. The current quantitative method using SID-SPE-DMS-MS provides throughput which is 7.5 to 30 times higher than that of LC-MS and provides a path to pre-clinical radiation dose estimation.Graphical Abstract [ABSTRACT FROM AUTHOR]

CAP256 is one of the highly potent, broadly neutralizing monoclonal antibodies (bNAb) designed for HIV-1 therapy. During the process development of one of the constructs, an unexpected product-related impurity was observed via microfluidics gel electrophoresis. A panel of complementary LC-MS analyses was applied for the comprehensive characterization of CAP256 which included the analysis of the intact and reduced protein, the middle-up approach, and a set of complementary peptide mapping techniques and verification of the disulfide bonds. The designed workflow allowed to identify a clip within a protruding acidic loop in the CDR-H3 region of the heavy chain, which can lead to the decrease of bNAb potency. This characterization explained the origin of the additional species reflected by the reducing gel profile. An intra-loop disulfide bond linking the two fragments was identified, which explained why the non-reducing capillary electrophoresis (CE) profile was not affected. The extensive characterization of CAP256 post-translational modifications was performed to investigate a possible cause of CE profile complexity and to illustrate other structural details related to this molecule’s biological function. Two sites of the engineered Tyr sulfation were verified in the antigen-binding loop, and pyroglutamate formation was used as a tool for monitoring the extent of antibody clipping. Overall, the comprehensive LC-MS study was crucial to (1) identify the impurity as sequence clipping, (2) pinpoint the clipping location and justify its susceptibility relative to the molecular structure, (3) lead to an upstream process optimization to mitigate product quality risk, and (4) ultimately re-engineer the sequence to be clip-resistant.ᅟ [ABSTRACT FROM AUTHOR]

Identification of the metabolites is an essential step in metabolomics study to interpret the regulatory mechanism of pathological and physiological processes. However, it is still difficult in LC-MSn-based studies because of the complexity of mass spectrometry, chemical diversity of metabolites, and deficiency of standards database. In this work, a comprehensive strategy is developed for accurate and batch metabolite identification in nontargeted metabolomics studies. First, a well-defined procedure was applied to generate reliable and standard LC-MS2 data, including tR, MS1, and MS2 information at a standard operational procedure. An in-house database including about 2000 metabolites was constructed and used to identify the metabolites in nontargeted metabolic profiling by retention time calibration using internal standards, precursor ion alignment and ion fusion, auto-MS2 information extraction and selection, and database batch searching and scoring. As an application example, a pooled serum sample was analyzed to deliver the strategy, and 202 metabolites were identified in the positive ion mode. It shows our strategy is useful for LC-MSn-based nontargeted metabolomics study. [ABSTRACT FROM AUTHOR]

High mobility group box 1 (HMGB1) is a nonhistone chromosomal protein, which can be secreted through a variety of pathways and bind to pattern recognition receptors to release pro-inflammatory cytokines. Previous studies have suggested that HMGB1 is upregulated in numerous inflammatory diseases and that it could be a biomarker for such diseases. However, these studies used immunoassay-based methods to analyze serum HMGB1. Autoantibodies to HMGB1 in serum are found in healthy control subjects as well as in patients with different diseases. HMGB1 also binds to haptoglobin, a highly abundant plasma protein. This means that antibodies used in immunoassays must compete with binding of HMGB1 to endogenous serum HMGB1 autoantibodies and haptoglobin. To overcome these potential problems, we developed and validated a specific and sensitive assay based on stable isotope dilution and immunopurification to quantify HMGB1 in plasma and serum using two-dimensional nano-ultra-high-performance liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry. Using this assay, we found that serum HMGB1 in 24 healthy control subjects (6.0 ± 2.1 ng/mL) was above the mean concentration reported for 18 different diseases (5.4 ± 2.8 ng/mL) where the analyses were conducted with immunoassay methodology. In light of our finding, the role of HMGB1 in these diseases will have to be re-evaluated. The concentration of HMGB1 in citrated and EDTA-treated plasma from the same healthy control subjects was below the limit of detection of our assay (1 ng/mL), confirming that HMGB1 in serum arises when blood is allowed to clot. This means that future studies on the role of HMGB1 in vivo should be conducted on plasma rather than serum. [ABSTRACT FROM AUTHOR]

Folate cofactors play a key role in one-carbon metabolism. Analysis of individual folate species is hampered by the low chemical stability and high interconvertibility of folates, which can lead to severe experimental bias. Here, we present a complete workflow that employs simultaneous extraction and stabilization of folates by derivatization. We perform reductive methylation employing stable isotope labeled reagents to retain information on the position and redox state of one-carbon units as well as the redox state of the pteridine ring. The derivatives are analyzed by a targeted LC(HILIC)-MS/MS method without the need for deconjugation, thereby also preserving the glutamation state of folates. The presented method does not only improve analyte coverage and sensitivity as compared to other published methods, it also greatly simplifies sample handling and storage. Finally, we report differences in the response of bacterial and mammalian systems to pharmacological inhibition of dihydrofolate reductase. [ABSTRACT FROM AUTHOR]

Therapeutic monoclonal antibodies (mAbs) are an important class of drugs for a wide spectrum of human diseases. Liquid chromatography (LC) coupled to mass spectrometry (MS) is one of the techniques in the forefront for comprehensive characterization of analytical attributes of mAbs. Among various protein chromatography modes, hydrophobic interaction chromatography (HIC) is a popular offline nondenaturing separation technique utilized to purify and analyze mAbs, typically with the use of non-MScompatible mobile phases. Herein we demonstrate for the first time, the application of direct HIC-MS and HIC-tandem MS (MS/MS) with electron capture dissociation (ECD) for analyzing intact mAbs on quadrupole-time-of-flight (Q-TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, respectively. Our method allows for rapid determination of relative hydrophobicity, intact masses, and glycosylation profiles of mAbs as well as sequence and structural characterization of the complementarity-determining regions in an online configuration. [ABSTRACT FROM AUTHOR]

This laboratory experiment employs an innovative approach to introduce undergraduates to liquid chromatography-tandem mass spectrometry (LC-MS/MS) by developing a method based on multiple reaction monitoring (MRM). During this qualitative investigation, students operated a triple quadrupole mass spectrometer in the MS scan and product ion scan modes to determine precursor and product ions, respectively, for a vanillin, ethyl vanillin, and coumarin standard mixture. After identifying precursor → product ion transitions, students conducted a 2 min liquid chromatography run in MRM mode. Students were astonished by the speed, selectivity, and sensitivity of the LC-MS/MS method compared to that of a liquid chromatograph with ultraviolet detection. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS2/Au-NP (gold nanoparticle)-L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS2 nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g-1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 μg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics. [ABSTRACT FROM AUTHOR]

Plasma stability assessment under physiological temperature is an essential step for developing and optimizing antibody drug conjugate (ADC) molecules, especially those with cleavable linkers. The assessment of plasma stability often requires monitoring multiple analytes using a combination of bioanalytical assays for free payloads, conjugated payloads (or conjugated antibodies), total antibodies, and payloads that have migrated from antibodies to plasma constituent proteins. Bioanalytical assays are needed in early drug discovery to quickly screen diverse ADC candidates of different antibody constructs, linker variants, and antibody anchor sites. To improve the sensitivity and selectivity of LC/MS/MS-based assays for the assessment, immunocapture has been widely used for extracting ADCs and unconjugated antibodies from plasma samples. In this study, a novel two-step immunocapture LC/MS/MS assay was described to allow the quantification of conjugated payloads, total antibodies, and migrated payloads forming adducts with albumin in the plasma samples for stability assessment. A target antigen immobilized on magnetic beads was used to exhaustively extract the ADC and antibody-associated species. The remaining supernatant was then extracted further with anti-albumin beads for recovering the albumin-associated adducts for quantification. The method was optimized for higher efficiency and cost-effectiveness using microwave enhanced papain-based enzymatic cleavage for measuring conjugated payloads of ADCs and lysyl endopeptidase cleavage in the total antibody assay. A maleimide linker-based ADC with a proprietary payload, TAK-001, was used to demonstrate the streamlined workflow of the ADC stability assessment. The method could provide valuable evaluation of the stability of the ADC as well as the quantitative assessment of the albumin adducts formed from the linker-payload migration in mouse and human plasma. Furthermore, the method should be readily adaptable for other ADCs using thiol-maleimide conjugation chemistry. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAb) and related molecules are being developed at a remarkable pace as new therapeutics for the treatment of diseases ranging from cancer to inflammatory disorders. However, characterization of these molecules at all stages of development and manufacturing presents tremendous challenges to existing analytical technologies because of their large size (ca. 150 kDa) and inherent heterogeneity resulting from complex glycosylation patterns and other post-translational modifications. Multidimensional liquid chromatography is emerging as a powerful platform technology that can be used to both improve analysis speed for these molecules by combining existing one-dimensional separations into a single method (e.g., Protein A affinity separation and size-exclusion chromatography) and increasing the resolving power of separations by moving from one dimension of separation to two. In the current study, we have demonstrated the ability to combine hydrophilic interaction (HILIC) and RP separations in an online comprehensive 2D separation coupled with high resolution MS detection (HILIC × RP-HRMS). We find that active solvent modulation (ASM) is critical for coupling these two separation modes, because it mitigates the otherwise serious negative impact of the acetonitrile-rich HILIC mobile phase on the second dimension RP separation. The chromatograms obtained from these HILIC × RP-HRMS separations of mAbs at the subunit level reveal the extent of glycosylation on the Fc/2 and Fd subunits in analysis times on the order of 2 h. In comparison to previous CEX × RP separations of the same molecules, we find that chromatograms from the HILIC × RP separations are richer and reveal separation of some glycoforms that coelute in the CEX × RP separations. [ABSTRACT FROM AUTHOR]

Carnitines play important roles in fatty acid oxidation and branched chain amino acid metabolism. The disturbance of acylcarnitines is associated with occurrence and development of many diseases. Comprehensive acylcarnitine identification can greatly benefit their targeted detection, following disease differential diagnosis and possible mechanism study. In this study, we developed a novel strategy to identify as many acylcarnitines as possible based on liquid chromatography-highresolution mass spectrometry (LC-HRMS). The layer-layer progressive strategy first integrated the initial full scan MS/data-dependent MS/MS monitoring (ddMS²) acquisition and the following parallel reaction monitoring (PRM) to analyze a pooled biological sample. Also 733 possible acylcarnitines were identified containing characteristic high-resolution MS/MS features. Further, accurate mass, retention rules, and HRMS/MS information were used to define subclasses and predict undetected acylcarnitine homologues in each subclass, leading to more acylcarnitines to our newly constructed database. As a result, 758 acylcarnitines were contained in the database, having exact mass, retention time, and MS/MS information, which is the most comprehensive list of acylcarnitines reported to date. Applying this database, 241, 515, and 222 acylcarnitines were rapidly and reliably annotated in human plasma, human urine, and rat liver tissue. This novel strategy enables large-scale identification of acylcarnitines, and a similar method can also be used for identification of other metabolites. [ABSTRACT FROM AUTHOR]

Due to its sensitivity and productivity, bottom-up proteomics based on liquid chromatography-mass spectrometry (LC-MS) has become the core approach in the field. The de facto standard LC-MS platform for proteomics operates at sub-µL/min flow rates, and nanospray is required for efficiently introducing peptides into a mass spectrometer. Although this is almost a "dogma", this view is being reconsidered in light of developments in highly efficient chromatographic columns, and especially with the introduction of exceptionally sensitive MS instruments. Although conventional-flow LC-MS platforms have recently penetrated targeted proteomics successfully, their possibilities in discovery-oriented proteomics have not yet been thoroughly explored. Our objective was to determine what are the extra costs and what optimization and adjustments to a conventional-flow LC-MS system must be undertaken to identify a comparable number of proteins as can be identified on a nanoLC-MS system. We demonstrate that the amount of a complex tryptic digest needed for comparable proteome coverage can be roughly 5-fold greater, providing the column dimensions are properly chosen, extra-column peak dispersion is minimized, column temperature and flow rate are set to levels appropriate for peptide separation, and the composition of mobile phases is fine-tuned. Indeed, we identified 2 835 proteins from 2 µg of HeLa cells tryptic digest separated during a 60 min gradient at 68 µL/min on a 1.0 mm x 250 mm column held at 55 °C and using an aqua-acetonitrile mobile phases containing 0.1% formic acid, 0.4% acetic acid, and 3% dimethyl sulfoxide. Our results document that conventional-flow LC-MS is an attractive alternative for bottom-up exploratory proteomics. [ABSTRACT FROM AUTHOR]

Separation and structural identification of lipids remain a major challenge for contemporary lipidomics. Regioisomeric lipids differing only in position(s) of unsaturation are often not differentiated by conventional liquid chromatography-mass spectrometry approaches leading to the incomplete, or sometimes incorrect, assignation of molecular structure. Here we describe an investigation of the gas phase separations by differential-mobility spectrometry (DMS) of a series of synthetic analogues of the recently described 1-deoxysphingosine. The dependence of the DMS behavior on the position of the carbon-carbon double bond within the ionized lipid is systematically explored and compared to trends from complementary investigations, including collision cross-sections measured by drift tube ion mobility, reaction efficiency with ozone, and molecular dynamics simulations. Consistent trends across these modes of interrogation point to the importance of direct, through-space interactions between the charge site and the carbon-carbon double bond. Differences in the geometry and energetics of this intramolecular interaction underpin DMS separations and influence reactivity trends between regioisomers. Importantly, the disruption and reformation of these intramolecular solvation interactions during DMS are proposed to be the causative factor in the observed separations of ionized lipids which are shown to have otherwise identical collision cross-sections. These findings provide key insights into the strengths and limitations of current ion-mobility technologies for lipid isomer separations and can thus guide a more systematic approach to improved analytical separations in lipidomics. [ABSTRACT FROM AUTHOR]

Bioanalysis of antibody-drug conjugates (ADCs) is challenging due to the complex, heterogeneous nature of their structures and their complicated catabolism. To fully describe the pharmacokinetics (PK) of an ADC, several analytes are commonly quantified, including total antibody, conjugate, and payload. Among them, conjugate is the most challenging to measure, because it requires detection of both small and large molecules as one entity. Existing approaches to quantify the conjugated species of ADCs involve a ligand binding assay (LBA) for conjugated antibody or hybrid LBA/liquid chromatography/tandem mass spectrometry (LC/MS/MS) for quantitation of conjugated drug. In our current work for a protein-drug conjugate (PDC) using the Centyrin scaffold, a similar concept to ADCs but with smaller protein size, an alternative method to quantify the conjugate by using a surrogate peptide approach, was utilized. The His-tagged proteins were isolated from biological samples using immobilized metal affinity chromatography (IMAC), followed by trypsin digestion. The tryptic peptide containing the linker attached to the payload was used as a surrogate of the conjugate and monitored by LC/MS/MS analysis. During method development and its application, we found that hydrolysis of the succinimide ring of the linker was ubiquitous, taking place at many stages during the lifetime of the PDC including in the initial drug product, in vivo in circulation in the animals, and ex vivo during the trypsin digestion step of the sample preparation. We have shown that hydrolysis during trypsin digestion is concentration-independent and consistent during the work flow--therefore, having no impact on assay performance. However, for samples that have undergone extensive hydrolysis prior to trypsin digestion, significant bias could be introduced if only the non-hydrolyzed form is considered in the quantitation. Therefore, it is important to incorporate succinimide hydrolysis products in the quantitation method in order to provide an accurate estimation of the total conjugate level. More importantly, the LC/MS/MS-based method described here provides a useful tool to quantitatively evaluate succinimide hydrolysis of ADCs in vivo, which has been previously reported to have significant impact on their stability, exposure, and efficacy. [ABSTRACT FROM AUTHOR]

Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging, because of the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (slow off-rate modified aptamer) is a novel reagent that binds, with high affinity, to a far distinct epitope of glypican-3, when compared to all available antibody reagents generated in-house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development, especially when traditional antibody reagents cannot meet assay requirements. [ABSTRACT FROM AUTHOR]

Ion mobility spectrometry enhances the performance characteristics of liquid chromatography-mass spectrometry workflows intended to steroid profiling by providing a new separation dimension and a novel characterization parameter, the so-called collision cross section (CCS). This work proposes the first CCS database for 300 steroids (i.e., endogenous, including phase I and phase II metabolites, and exogenous synthetic compounds), which involves 1080 ions and covers the CCS of 127 androgens, 84 estrogens, 50 corticosteroids, and 39 progestagens. This large database provides information related to all the ionized species identified for each steroid in positive electrospray ionization mode as well as for estrogens in negative ionization mode. CCS values have been measured using nitrogen as drift gas in the ion mobility cell. Generally, direct correlation exists between mass-to-charge ratio (m/z) and CCS because both are related parameters. However, several steroids mainly steroid glucuronides and steroid esters have been characterized as more compact or elongated molecules than expected. In such cases, CCS results in additional relevant information to retention time and mass spectral data for the identification of steroids. Moreover, several isomeric steroid pairs (e.g., 5β-androstane-3,17-dione and 5α-androstane-3,17-dione) have been separated based on their CCS differences. These results indicate that adding the CCS to databases in analytical workflows increases selectivity, thus improving the confidence in steroids analysis. Consequences in terms of identification and quantification are discussed. Quality criteria and a construction of an interlaboratory reproducibility approach are also reported for the obtained CCS values. The CCS database described here is made publicly available. [ABSTRACT FROM AUTHOR]

Chemical exposure in household dust poses potential risks to human health but has been studied incompletely thus far. Most analytical studies have focused on one or several compound classes, with analysis performed by either liquid or gas chromatography coupled with mass spectrometry (LC-MS or GC-MS). However, a comprehensive investigation of individual dust samples is missing. The present study comprehensively characterizes chemicals in dust by applying a combination of target, suspect, and nontarget screening approaches using both LC and GC with quadrupole time-of-flight (Q/TOF) MS. First, the extraction method was optimized to streamline detection of LC-Q/TOF and GC-Q/TOF amenable compounds and was successfully validated with over 100 target compounds. Nontarget screening with GC-Q/TOF was done by spectral deconvolution followed by a library search. Suspect screening by LC-Q/TOF was carried out with an accurate mass spectral library. Finally, LC-Q/TOF nontarget screening was carried out by extracting molecular features, acquiring tandem mass spectrometric (MS/MS) spectra, and performing compound identification by use of in silico fragmentation software tools. In total, 271 chemicals could be detected in 38 dust samples, 163 of which could be unambiguously confirmed by a reference standard. Many of them, such as the plastic leachable 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione (CAS 82304-66-3) and three organofluorine compounds, are of emerging concern and their presence in dust has been underestimated. Advantages and drawbacks of the different approaches and analytical instruments are critically discussed. [ABSTRACT FROM AUTHOR]

Gut microbiota plays important roles in the host health. The host and symbiotic gut microbiota coproduce a large number of metabolites during the metabolism of food and xenobiotics. The analysis of fecal metabolites can provide a noninvasive manner to study the outcome of the host-gut microbiota interaction. Herein, we reported the comprehensive profiling of fecal metabolome of mice by an integrated chemical isotope labeling combined with liquid chromatography-mass spectrometry (CIL-LC-MS) analysis. The metabolites are categorized into several submetabolomes based on the functional moieties (i.e., carboxyl, carbonyl, amine, and thiol) and then analysis of the individual submetabolome was performed. The combined data from the submetabolome form the metabolome with relatively high coverage. To this end, we synthesized stable isotope labeling reagents to label metabolites with different groups, including carboxyl, carbonyl, amine, and thiol groups. We detected 2302 potential metabolites, among which, 1388 could be positively or putatively identified in feces of mice. We then further confirmed 308 metabolites based on our established library of chemically labeled standards and tandem mass spectrometry analysis. With the identified metabolites in feces of mice, we established mice fecal metabolome database, which can be used to readily identify metabolites from feces of mice. Furthermore, we discovered 211 fecal metabolites exhibited significant difference between Alzheimer's disease (AD) model mice and wild type (WT) mice, which suggests the close correlation between the fecal metabolites and AD pathology and provides new potential biomarkers for the diagnosis of AD. [ABSTRACT FROM AUTHOR]

Dried blood spots (DBS), collected for newborn screening programs in the United States, have been used to screen for congenital metabolic diseases in newborns for over 50 years. DBS provide an easy and inexpensive way to collect and store peripheral blood specimens and present an excellent resource for studies on the assessment of chemical exposures in newborns. In this study, a selective and sensitive method was developed for the analysis of iodide in DBS by high performance liquid chromatography electrospray tandem mass spectrometry. Accuracy, inter- and intraday precision, matrix effects, and detection limits of the method were determined. Further validation of the method was accomplished by concurrent analysis of whole blood and fortified blood spotted on a Whatman 903 filter card. A significant positive correlation was found between measured concentrations of iodide in venous whole blood and the same blood spotted as DBS. The method limit of detection was 0.15 ng/mL iodide. The method was further validated by the analysis of a whole blood sample certified for iodide levels (proficiency testing sample) by spotting on a filter card. Twenty DBS samples collected from newborns in New York State were analyzed to demonstrate the applicability of the method. The measured concentrations of iodide in whole blood of newborns from New York State ranged between

ABSTRACT: One unusual and challenging scientific field that has received only cursory attention to date is the threedimensional (3D) microstructure and spatial distribution of drug(s) and formulation materials in solid dosage forms. This study aims to provide deeper insight into the relationships between the microstructure of multiple-unit pellet system (MUPS) tablets and the spatial distribution of the active pharmaceutical ingredient (API) and excipients to facilitate the design of quantitative models for drug delivery systems. Synchrotron radiation X-ray microcomputed tomography (SR-μCT) was established as a 3D structure elucidation technique, which, in conjunction with liquid chromatography coupled to mass spectrometry (LC-MS) or liquid chromatography with evaporative light-scattering detector (LC-ELSD) enables chemical analysis of tablets. On the basis of the specific interior construction of theophylline MUPS tablets, the spatial distribution of materials was acquired by quantifying microregion samples that had been validated by SR-μCT for their locations in the MUPS tablets. The 3D structure of the MUPS tablets was catalogued as three structural domains: a matrix layer (ML), a protective cushion layer (PCL), and pellets (PL). Compared with the components in the ML, components in the PL had a larger proportion of theophylline, sucrose, and diethyl phthalate and a smaller proportion of lactose and sodium lauryl sulfate, whereas glyceryl monostearate was found to account for a large portion of the PCL. Microstructural characterization-guided zonal chemical determination represents a new approach for quality assessment and the development of drug delivery systems with in-depth insight into their constituent layers on a new scale. [ABSTRACT FROM AUTHOR]

In this Article, we have reported a fully automated online method to carry out proteolysis and glycopeptide enrichment in sequence for nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) analysis. By implementing two serial thermoresponsive porous polymer membrane reactors (TPPMRs), in which the TPPM could be immobilized either with trypsin for proteolysis or with lectins for glycopeptide enrichment, the entire pretreatment procedure can be performed online in about an hour. The TPPM was fabricated by coating polystyrene-maleic anhydride-Nisopropylacrylamide (PS-MAn-PNIPAm), which was synthesized by reversible addition-fragmentation chain transfer polymerization, on a Nylon sheet. Because of the thermoresponsive nature of PNIPAm, it formed micelle cavities and changed its morphology at elevated temperatures, resulting in enhanced interactions between the enzyme or lectins and the proteins/ peptides flowing through the membrane. The performances of the TPPMs were evaluated by varying the temperature conditions and the amount of standard proteins, showing that both proteolysis and glycopeptide enrichment with online deglycosylation were highly efficient at 37 °C. The developed online serial TPPMRs-nLC-ESI-MS/MS method was applied to the human plasma sample (1.5 μL) and a total of 262 N-glycopeptides could be identified from 155 glycoproteins. Thus, the present work demonstrates a fully automated high speed analytical protocol for online proteolysis and glycopeptide enrichment, which is extremely useful for analyzing small amounts of the proteome samples. [ABSTRACT FROM AUTHOR]

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer "middle-down" protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography−tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis. [ABSTRACT FROM AUTHOR]

Abnormal expression of C-terminal p53 isoforms α, ß, and γ can cause the development of cancers including breast cancer. To date, much evidence has demonstrated that these isoforms can differentially regulate target genes and modulate their expression. Thus, quantification of individual isoforms may help to link clinical outcome to p53 status and to improve cancer patient treatment. However, there are few studies on accurate determination of p53 isoforms, probably due to sequence homology of these isoforms and also their low abundance. In this study, a targeted proteomics assay combining molecularly imprinted polymers (MIPs) and liquid chromatography-tandem mass spectrometry (LCMS/ MS) was developed for simultaneous quantification of C-terminal p53 isoforms. Isoform-specific surrogate peptides (i.e., KPLDGEYFTLQIR (peptide-α) for isoform α, KPLDGEYFTLQDQTSFQK (peptide-ß) for isoform ß, and KPLDGEYFTLQMLLDLR (peptide-γ) for isoform γ) were first selected and used in both MIPs enrichment and mass spectrometric detection. The common sequence KPLDGEYFTLQ of these three surrogate peptides was used as single template in MIPs. In addition to optimization of imprinting conditions and characterization of the prepared MIPs, binding affinity and cross-reactivity of the MIPs for each surrogate peptide were also evaluated. As a result, a LOQ of 5 nM was achieved, which was >15-fold more sensitive than that without MIPs. Finally, the assay was validated and applied to simultaneous quantitative analysis of C-terminal p53 isoforms α, ß, and γ in several human breast cell lines (i.e., MCF- 10A normal cells, MCF-7 and MDA-MB-231 cancer cells, and drug-resistant MCF-7/ADR cancer cells). This study is among the first to employ single template MIPs and cross-reactivity phenomenon to select isoform-specific surrogate peptides and enable simultaneous quantification of protein isoforms in LC-MS/MS-based targeted proteomics. [ABSTRACT FROM AUTHOR]

Gangliosides are sialic acid-containing glycosphingolipids recognized to play essential role in biological processes. Both the glycan and lipid structures influence their biological function and thus necessitate their determination as intact molecular species. To our knowledge, no multiplexed method for intact gangliosides currently exists. In this paper, we aimed to demonstrate an approach for isobaric labeling of intact gangliosides. Specifically, we carried out the rapid, chemoselective oxidation of sialic acid side chain in common ganglioside core structures using NaIO[sub 4] followed by ligation with a carbonyl-reactive isobaric tandem mass tag (TMT) reagent and subsequent RPLC-MS/MS analysis. Attachment of the isobaric label was observed to improve the ionization efficiency of complex gangliosides using electrospray ionization. Fragmentation of the resulting [M + 2H]2+ ions of TMT-labeled gangliosides provided information-rich spectra containing fragments from the glycan, lipid, and TMT reporter ions. This facile approach enabled simultaneous quantification of up to six samples as well as identification of glycan and lipid compositions in a single injection. As a proof-of-concept, using porcine brain total ganglioside extracts pooled at known ratios, we obtained overall sample-to-sample precision of <12% RSD and mean error of <10%. This showcased the great promise and feasibility of this strategy for high-throughput analysis of intact gangliosides in biological extracts. [ABSTRACT FROM AUTHOR]

Evidence for the existence of condensed-phase isomers of silver-lipoate clusters, Ag29(LA)12, where LA = (R)-a lipoic acid, was obtained by reversed-phase ion-pair liquid chromatography with in-line UV-vis and electro-spray ionization (ESI)-MS detection. All components of a raw mixture were separated according to surface chemistry and increasing size via reversed-phase gradient HPLC methods and identified by their corresponding m/z ratio by ESI in the negative ionization mode. Aqueous and methanol mobile-phase mixtures, each containing 400 mM hexafluoroisopropanol (HFIP)-15 mM triethylamine (TEA), were employed to facilitate the interaction between the clusters and stationary phase via formation of ion-pairs. TEA-HFIP (triethylammonium-hexafluoroisoprop-oxide) had been shown to provide superior chromatographic peak shape and mass spectral signal compared with alternative modifiers such as TEAA (triethylammo-nium-acetate) for analysis of oligonucleotide samples. Liquid chromatographic separation prior to mass spectrometry detection facilitated sample analysis by production of simplified mass spectra for each eluting cluster species and provided insight into the existence of at least two major solution-phase isomers of Ag29(LA)12. UV-vis detection in-line with ESI analysis provided independent confirmation of the existence of the isomers and their similar electronic structure as judged from their identical optical spectra in the 300-500 nm range. [Diastereomerism provides a possible interpretation for the near-equal abundance of the two forms, based on a structurally defined nonaqueous homologue.] [ABSTRACT FROM AUTHOR]

S-Nitrosothiols (RSNOs) constitute a circulating endogenous reservoir of nitric oxide and have important biological activities. In this study, an online coupling of solid-phase derivatization (SPD) with liquid chromatography-mass spectrometry (LC-MS) was developed and applied in the analysis of low-molecular-mass RSNOs. A derivatizing-reagent-modified polymer monolithic column was prepared and adapted for online SPD-LC-MS. Analytes from the LC autosampler flowed through the monolithic column for derivatization and then directly into the LC-MS for analysis. This integration of the online derivatization, LC separation, and MS detection facilitated system automation, allowing rapid, laborsaving, and sensitive detection of RSNOs. S-Nitro-soglutathione (GSNO) was quantified using this automated online method with good linearity (R2 = 0.9994); the limit of detection was 0.015 nM. The online SPD-LC-MS method has been used to determine GSNO levels in mouse samples, 138 ± 13.2 nM of endogenous GSNO was detected in mouse plasma. Besides, the GSNO concentrations in liver (64.8 ± 11.3 pmol/mg protein), kidney (47.2 ± 6.1 pmol/mg protein), heart (8.9 ± 1.8 pmol/mg protein), muscle (1.9 ± 0.3 pmol/mg protein), hippocampus (5.3 ± 0.9 pmol/mg protein), striatum (6.7 ± 0.6 pmol/mg protein), cerebellum (31.4 ± 6.5 pmol/mg protein), and cortex (47.9 ± 4.6 pmol/mg protein) were also successfully quantified. When the derivatization was performed within 8 min, followed by LC-MS detection, samples could be rapidly analyzed compared with the offline manual method. Other low-molecular-mass RSNOs, such as S-nitrosocysteine and Snitrosocysteinylglycine, were captured by rapid precursor-ion scanning, showing that the proposed method is a potentially powerful tool for capture, identification, and quantification of RSNOs in biological samples. [ABSTRACT FROM AUTHOR]

The increasing focus on lipid metabolism has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids can exist as any one of a large number of double bond positional isomers, which are indistinguishable by single-stage mass spectrometry alone. Ozonolysis reactions coupled to mass spectrometry have previously been demonstrated as a means for localizing double bonds in unsaturated lipids. Here we describe an online, solution-phase reactor using ozone produced via a low-pressure mercury lamp, which generates aldehyde products diagnostic of cleavage at a particular double bond position. This flow-cell device is utilized in conjunction with structurally selective ion mobility-mass spectrometry. The lamp-mediated reaction was found to be effective for multiple lipid species in both positive and negative ionization modes, and the conversion efficiency from precursor to product ions was tunable across a wide range (20-95%) by varying the flow rate through the ozonolysis device. Ion mobility separation of the ozonolysis products generated additional structural information and revealed the presence of saturated species in a complex mixture. The method presented here is simple, robust, and readily coupled to existing instrument platforms with minimal modifications necessary. For these reasons, application to standard lipidomic workflows is possible and aids in more comprehensive structural characterization of a myriad of lipid species. [ABSTRACT FROM AUTHOR]

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-μLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by μLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the μLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics. [ABSTRACT FROM AUTHOR]

ABSTRACT: Replacement of capillary with solid substrates for sample loading and ionization has created many new possibilities for electrospray ionization mass spectrometry (ESI-MS). Surface modification is an attractive strategy to enhance the analytical capability of solid-substrate ESI-MS and allow understanding the relationship between surface activity of solid substrates and analytical properties. In this study, we performed surface modification of wooden tips with hydrophobic (-C18), basic (-NH2), and acidic (-SO3H) functional groups and applied various sampling methods, i.e., extractive sampling and direct loading, to comprehensively investigate the analytical properties of solid-substrate ESI-MS. Our results showed that, for the direct loading method, analytes with weak interactions with solid-substrate surface could be readily sprayed out for detection. While for the extractive sampling method, analytes strongly retained on solid-substrate surface could be selectively enriched and detected, and a washing step after sample loading could effectively remove unbound components for reducing interference. Overall, the insights on the effects of surface -- analyte interactions on the analytical features obtained in this study could aid the development of surface-modified strategies for enhancing the analytical capability of solid-substrate ESI-MS. [ABSTRACT FROM AUTHOR]

Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS3)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community, we developed a workflow that integrates the established software tools MaxQuant for spectra preprocessing, Xi for cross-linked peptide identification, and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 124 were subsequently quantified across 10 analyses with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides.ᅟ [ABSTRACT FROM AUTHOR]

The goal of metabolomics is to measure the entire range of small organic molecules in biological samples. In liquid chromatography-mass spectrometry-based metab-olomics, formidable analytical challenges remain in removing the nonbiological factors that affect chromatographic peak areas. These factors include sample matrix-induced ion suppression, chromatographic quality, and analytical drift. The combination of these factors is referred to as obscuring variation. Some metabolomics samples can exhibit intense obscuring variation due to matrix-induced ion suppression, rendering large amounts of data unreliable and difficult to interpret. Existing normalization techniques have limited applicability to these sample types. Here we present a data normalization method to minimize the effects of obscuring variation. We normalize peak areas using a batch-specific normalization process, which matches measured metabolites with isotope-labeled internal standards that behave similarly during the analysis. This method, called best-matched internal standard (B-MIS) normalization, can be applied to targeted or untargeted metabolomics data sets and yields relative concentrations. We evaluate and demonstrate the utility of B-MIS normalization using marine environmental samples and laboratory grown cultures of phytoplankton. In untargeted analyses, B-MIS normalization allowed for inclusion of mass features in downstream analyses that would have been considered unreliable without normalization due to obscuring variation. B-MIS normalization for targeted or untargeted metabolomics is freely available at https://github.com/IngallsLabUW/B-MIS-normalization. [ABSTRACT FROM AUTHOR]

Profiling of complex proteins by means of mass spectrometry (MS) frequently requires that certain chemical modifications of their covalent structure (e.g., reduction of disulfide bonds), be carried out prior to the MS or MS/MS analysis. Traditionally, these chemical reactions take place in the off-line mode to allow the excess reagents (the majority of which interfere with the MS measurements and degrade the analytical signal) to be removed from the protein solution prior to MS measurements. In addition to a significant increase in the analysis time, chemical reactions may result in a partial or full loss of the protein if the modifications adversely affect its stability, e.g,, making it prone to aggregation. In this work we present a new approach to solving this problem by carrying out the chemical reactions online using the reactive chromatography scheme on a size exclusion chromatography (SEC) platform with MS detection. This is achieved by using a cross-path reaction scheme, i.e., by delaying the protein injection onto the SEC column (with respect to the injection of the reagent plug containing a disulfide-reducing agent), which allows the chemical reactions to be carried out inside the column for a limited (and precisely controlled) period of time, while the two plugs overlap inside the column. The reduced protein elutes separately from the unconsumed reagents, allowing the signal suppression in ESI to be avoided and enabling sensitive MS detection. The new method is used to measure fucosylation levels of a plasma protein haptoglobin at the whole protein level following online reduction of disulfide-linked tetrameric species to monomeric units. The feasibility of top-down fragmentation of disulfide-containing proteins is also demonstrated using β2-microglobulin and a monoclonal antibody (mAb). The new online technique is both robust and versatile, as the cross-path scheme can be readily expanded to include multiple reactions in a single experiment (as demonstrated in this work by oxidatively labeling mAb on the column, followed by reduction of its disulfide bonds and MS analysis of the extent of oxidation within each chain of the molecule). [ABSTRACT FROM AUTHOR]

Glycated proteins are emerging as good indicators for diabetes and age related diseases. However, the platform for analysis of glycated proteome has been relatively less well established. We here introduce an online 2D-LC-HCD-MS/MS platform for comprehensive glycated peptide quantification. This platform includes a boronate affinity column in the first dimension for enrichment, reversed phase nanoLC column in the second dimension for separation, a benchtop Orbitrap mass spectrometer with HCD-MS/MS for peptide sequencing, and MaxQuant bioinformatics tool for identification and quantification of glycated peptides. This online 2D-LC-HCD-MS/MS platform has high enrichment efficiency with 85% of identified peptides in the enriched fraction as glycated, high sensitivity for detection of glycated peptides with LOD and LOQ at 1.2 and 2.4 pg, respectively, and high reproducibility with interday CVs < 20% for 80% of the glycated peptides. The number of glycated peptides quantified in in vitro glycated human plasma increased more than 3-fold using this platform in comparison to that obtained using 1D-LC-HCD-MS/MS platform without boronate affinity enrichment. Application of this online platform to human plasma identified 376 glycated peptides from 10 μg of protein digests. This highly sensitive and reproducible online 2D platform is promising for glycated protein analysis of complex clinical samples. [ABSTRACT FROM AUTHOR]

Monitoring human exposure to pesticides and pesticide residues (PRs) remains crucial for informing public health policies, despite strict regulation of plant protection product and biocide use. We used 72 low-cost silicone wristbands as noninvasive passive samplers to assess cumulative 5-day exposure of 30 individuals to polar PRs. Ethyl acetate extraction and LC-MS/MS analysis were used for the identification of PRs. Thirty-one PRs were detected of which 15 PRs (48%) were detected only in worn wristbands, not in environmental controls. The PRs included 16 fungicides (52%), 8 insecticides (26%), 2 herbicides (6%), 3 pesticide derivatives (10%), 1 insect repellent (3%), and 1 pesticide synergist (3%). Five detected pesticides were not approved for plant protection use in the EU. Smoking and dietary habits that favor vegetable consumption were associated with higher numbers and higher cumulative concentrations of PRs in wristbands. Wristbands featured unique PR combinations. Our results suggest both environment and diet contributed to PR exposure in our study group. Silicone wristbands could serve as sensitive passive samplers to screen population-wide cumulative dietary and environmental exposure to authorized, unauthorized and banned pesticides. [ABSTRACT FROM AUTHOR]

Among all the nuclear-receptor mediated endocrine disruptive effects, antiandrogenicity is frequently observed in aquatic environments and may pose a risk to aquatic organisms. Linking these effects to responsible chemicals is challenging and a great share of antiandrogenic activity detected in the environment has not been explained yet. To identify drivers of this effect at a hot spot of antiandrogenicity in the German river Holtemme, we applied effect-directed analysis (EDA) including a parallel fractionation approach, a downscaled luciferase reporter gene cell-based anti-AR-CALUX assay and LC-HRMS/MS nontarget screening. We identified and confirmed the highly potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and two derivatives in the active fractions. The relative potency of C47 to the reference compound flutamide was over 5.2, whereas the derivatives were less potent. C47 was detected at a concentration of 13.7 μg/L, equal to 71.4 ±g flutamide equivalents per liter (FEq/L) in the nonconcentrated water extract that was posing an antiandrogenic activity equal to 45.5 (±13.7 SD) FEq/L. Thus, C47 was quantitatively confirmed as the major cause of the measured effect in vitro. Finally, the antiandrogenic activity of C47 and one derivate was confirmed in vivo in spiggin-gfp Medaka. An endocrine disrupting effect of C47 was observed already at the concentration equal to the concentration in the nonconcentrated water extract, underlining the high risk posed by this compound to the aquatic ecosystem. This is of some concern since C47 is used in a number of consumer products indicating environmental as well as human exposure. [ABSTRACT FROM AUTHOR]