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Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.

Cyclic peptides are an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guidance on further structure optimization. However, the unambiguous characterization of cyclic peptide soft spots remains a significant challenge during early stage discovery studies, as amide bond hydrolysis forms a linearized isobaric sequence with the addition of a water molecule, regardless of the amide hydrolysis location. In this study, an innovative strategy was developed to enable the rapid and definitive identification of cyclic peptide soft spots by isotope-labeled reductive dimethylation and mass spectrometry fragmentation. The dimethylated immonium ion with enhanced MS signal at a distinctive m / z in MS/MS fragmentation spectra reveals the N-terminal amino acid on a linearized peptide sequence definitively and, thus, significantly simplifies the soft spot identification workflow. This approach has been evaluated to demonstrate the potential of isotope-labeled dimethylation to be a powerful analytical tool in cyclic peptide drug discovery and development.

Over the past three decades, mass spectrometry imaging (MSI) has emerged as a valuable tool for the spatial localization of drugs and metabolites directly from tissue surfaces without the need for labels. MSI offers molecular specificity, making it increasingly popular in the pharmaceutical industry compared to conventional imaging techniques like quantitative whole-body autoradiography (QWBA) and immunohistochemistry, which are unable to distinguish parent drugs from metabolites. Across the industry, there has been a consistent uptake in the utilization of MSI to investigate drug and metabolite distribution patterns, and the integration of MSI with omics technologies in preclinical investigations. To continue the further adoption of MSI in drug discovery and development, we believe there are two key areas that need to be addressed. First, there is a need for accurate quantification of analytes from MSI distribution studies. Second, there is a need for increased interactions with regulatory agencies for guidance on the utility and incorporation of MSI techniques in regulatory filings. Ongoing efforts are being made to address these areas, and it is hoped that MSI will gain broader utilization within the industry, thereby becoming a critical ingredient in driving drug discovery and development., (© 2024 John Wiley & Sons Ltd.)

The discovery of peptide therapeutics represents a fast-growing segment of pharmaceutical research. During the early discovery process, a large number of peptide candidates needs to be rapidly screened for metabolic stability in relevant biological matrices. In most cases, peptide stability assays are quantified using LC-MS/MS, which may take hours to analyze 384 samples and generates liters of solvent waste. Herein, we introduce a high-throughput screening (HTS) platform for peptide stability assessment founded on Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS). Full automation has been implemented for sample preparation with minimal manual intervention. The limit of detection, linearity, and reproducibility of the platform were evaluated, and metabolic stabilities have been determined for a number of peptide candidates. The MALDI-MS-based HTS workflow is able to analyze 384 samples in less than 1 h while only using 115 μL of total solvent. Although this process allows for very rapid assessment of peptide stability, given the nature of the MALDI process, it is noteworthy that spot-to-spot variations and ionization bias are observed. Therefore, LC-MS/MS may still be needed for confident, quantitative measurements and/or when the ionization efficiency of certain peptides is inadequate using MALDI.

Glucuronidation is a common phase II metabolic process for drugs and xenobiotics which increases their solubility for excretion. Acyl glucuronides (glucuronides of carboxylic acids) present concerns as they have been implicated in gastrointestinal toxicity and hepatic failure. Despite the substantial success in the bulk analysis of these species, previous attempts using traditional mass spectrometry imaging (MSI) techniques have completely or partially failed and therefore little is known about their localization in tissues. Herein, we use nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI), an ambient liquid extraction-based ionization technique, as a viable alternative to other MSI techniques to examine the localization of diclofenac, a widely used nonsteroidal anti-inflammatory drug, and its metabolites in mouse kidney and liver tissues. MSI data acquired over a broad m/z range showed low signals of the drug and its metabolites resulting from the low ionization efficiency and substantial signal suppression on the tissue. Significant improvements in the signal-to-noise were obtained using selected ion monitoring (SIM) with m/z windows centered around the low-abundance ions of interest. Using nano-DESI MSI in SIM mode, we observed that diclofenac acyl glucuronide and hydroxydiclofenac are localized to the inner medulla and cortex of the kidney, respectively, which is consistent with the previously reported localization of enzymes that process diclofenac into its respective metabolites. In contrast, a uniform distribution of diclofenac and its metabolites was observed in the liver tissue. Concentration ratios of diclofenac and hydroxydiclofenac calculated from nano-DESI MSI data are generally in agreement to those obtained using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Collectively, our results demonstrate that nano-DESI MSI can be successfully used to image diclofenac and its primary metabolites and derive relative quantitative data from different tissue regions. Our approach will enable a better understanding of metabolic processes associated with diclofenac and other drugs that are difficult to analyze using commercially available MSI platforms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Acyl glucuronide (AG) metabolites of carboxylic acid-containing drugs and products of their transformations have long been implicated in drug-induced liver injury (DILI). To inform on the DILI risk arising from AG reactive intermediates, a comprehensive mechanistic study of enzyme-independent AG rearrangements using nuclear magnetic resonance (NMR) and density functional theory (DFT) was undertaken. NMR spectroscopy was utilized for structure elucidation and kinetics measurements of nine rearrangement and hydrolysis products of 1β-O-acyl glucuronide of ibufenac. To extract rate constants of rearrangement, mutarotation, and hydrolysis from kinetic data, 11 different kinetic models were examined. Model selection and estimated rate constant verification were supported by measurements of H/D kinetic isotope effects. DFT calculations of ground and transition states supported the proposed kinetic mechanisms and helped to explain the unusually fast intramolecular transacylation rates found for some of the intermediates. The findings of the current study reinforce the notion that the short half-life of parent AG and slow hydrolysis rates of AG rearrangement products are the two key factors that can influence the in vivo toxicity of AGs.

dropletProbe mass spectrometry is a novel technique for molecular characterization of surfaces. It can be used for rapid ex vivo analysis of therapeutics from thin animal tissue sections and has been shown to improve understanding of a drug's absorption, distribution, metabolism and excretion (ADME) properties. Here, we describe the tissue distribution analysis of diclofenac from a dosed whole-body mouse thin tissue section using a dropletProbe mass spectrometry system., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bruton's tyrosine kinase (BTK) is an essential node on the BCR signaling in B cells, which are clinically validated to play a critical role in B-cell lymphomas and various auto-immune diseases such as Multiple Sclerosis (MS), Pemphigus, and rheumatoid arthritis (RA). Although non-selective irreversible BTK inhibitors have been approved for oncology, due to the emergence of drug resistance in B-cell lymphoma associated with covalent inhibitor, there an unmet medical need to identify reversible, selective, potent BTK inhibitor as viable therapeutics for patients. Herein, we describe the identification of Hits and subsequence optimization to improve the physicochemical properties, potency and kinome selectivity leading to the discovery of a novel class of BTK inhibitors. Utilizing Met ID and structure base design inhibitors were synthesized with increased in vivo metabolic stability and oral exposure in rodents suitable for advancing to lead optimization., (Copyright © 2021. Published by Elsevier Ltd.)

dropletProbe mass spectrometry (MS) is an emerging tool for the rapid ex vivo analysis of drugs in tissues and whole-body sections. Its use has been demonstrated to better understand a drug's absorption, distribution, metabolism, and excretion (ADME) properties. To further optimize the overall utility of this technique, it is important to characterize and understand the various tissue matrix effects and extraction solvents on the overall performance of dropletProbe MS analyses. Herein, we systematically evaluated the impact of extraction solvents and various tissues on the relative detected signal intensities of a test set of diverse drugs. It was observed that the tissue matrix had a minimal effect on the performance of dropletProbe MS for the limited set of tested compounds once an optimized extraction solvent was identified. A general starting extraction solvent of 1:1 acetonitrile/water (v:v) was identified to efficiently extract the test set of compounds from various tissues. Next, the optimized conditions were used to map the distribution of the drug diclofenac and its metabolites in whole-body mouse sections. The relative tissue distribution of diclofenac and its metabolites, including the phase II acyl-glucuronide metabolite, were successfully determined with the technique. It is recommended these conditions are used as a general guideline when initiating dropletProbe MS studies of therapeutic drug-like compounds.

Peptides have become a fast-growing segment of the pharmaceutical industry over the past few decades. It is essential to develop cutting edge analytical techniques to support the discovery and development of peptide therapeutics, especially to examine their absorption, distribution, metabolism and excretion (ADME) properties. Herein, we utilized two label-free mass spectrometry (MS) based techniques to investigate representative challenges in developing therapeutic peptides, such as tissue distribution, metabolic stability and clearance. A tool proof-of-concept cyclic peptide, melanotan II, was used in this study. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which is a well-developed label-free imaging technique, was used to map the detailed molecular distribution of melanotan II and its metabolites. Droplet-based liquid microjunction surface sampling liquid chromatography-high resolution mass spectrometry (LMJ-SSP-LC-HRMS) was used in combination with MALDI-MSI to rapidly profile molecular information and provide structural insights on drug and metabolites. Using both techniques in parallel allowed a more comprehensive and complementary data set than using either technique independently. We envision MALDI-MSI and droplet-based LMJ-SSP-LC-HRMS, which can be used in combination or as standalone techniques, to become valuable tools for assessing the in vivo fate of peptide therapeutics in support of drug discovery and development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

As metabolism impacts the efficacy and safety of therapeutic peptides and proteins (TPPs), understanding of the metabolic fate of TPPs is critical for their preclinical and clinical development. Despite the continued increase of new TPPs entering clinical trials, the metabolite identification (MetID) of these emerging modalities remains challenging. In the present study, we report an analytical workflow for MetID of TPPs. Using insulin detemir as an example, we demonstrated that top-down differential mass spectrometry (dMS) was able to distinguish and discover metabolites from complex biological matrices. For structural interpretation, we developed an algorithm to generate a complete and nonredundant theoretical metabolite database for a TPP of any topology (e.g., branched, multicyclic, etc.). Candidate structures of a metabolite were obtained by matching the monoisotopic mass of a dMS feature to the theoretical metabolite database. Finally, the MS/MS sequence tags enabled unambiguous characterization of metabolite structures when isobaric/isomeric candidates were present. This platform is widely applicable to TPPs with complex structures and will ultimately guide the structural optimization of TPPs in pharmaceutical development.

Glucuronidation, a common phase II biotransformation reaction, is one of the major in vitro and in vivo metabolism pathways of xenobiotics. In this process, glucuronic acid is conjugated to a drug or a drug metabolite via a carboxylic acid, a hydroxy, or an amino group to form acyl-, O -, and/or N -glucuronide metabolites, respectively. This process is traditionally thought to be a detoxification pathway. However, some acyl-glucuronides react with biomolecules in vivo , which may result in immune-mediated idiosyncratic drug toxicity (IDT). In order to avoid this, one may attempt in early drug discovery to modify the lead compounds in such a manner that they then have a lower probability of forming reactive acyl-glucuronide metabolites. Because most drugs or drug candidates bear multiple functionalities, e.g., hydroxy, amino, and carboxylic acid groups, glucuronidation can occur at any of those. However, differentiation of isomeric acyl-, N- , and O -glucuronide derivatives of drugs is challenging. In this study, gas-phase ion-molecule reactions between deprotonated glucuronide metabolites and BF 3 followed by collision-activated dissociation (CAD) in a linear quadrupole ion trap mass spectrometer were demonstrated to enable the differentiation of acyl-, N- , and O -glucuronides. Only deprotonated N -glucuronides and deprotonated, migrated acyl-glucuronides form the two diagnostic product ions: a BF 3 adduct that has lost two HF molecules, [M - H + BF 3 - 2HF] - , and an adduct formed with two BF 3 molecules that has lost three HF molecules, [M - H + 2BF 3 - 3HF] - . These product ions were not observed for deprotonated O -glucuronides and unmigrated, deprotonated acyl-glucuronides. Upon CAD of the [M - H + 2BF 3 - 3HF] - product ion, a diagnostic fragment ion is formed via the loss of 2-fluoro-1,3,2-dioxaborale (MW of 88 Da) only in the case of deprotonated, migrated acyl-glucuronides. Therefore, this method can be used to unambiguously differentiate acyl-, N- , and O -glucuronides. Further, coupling this methodology with HPLC enables the differentiation of unmigrated 1-β-acyl-glucuronides from the isomeric acyl-glucuronides formed upon acyl migration. Quantum chemical calculations at the M06-2X/6-311++G(d,p) level of theory were employed to probe the mechanisms of the reactions of interest.

Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous clinical trials have been reported using covalent inhibitors to target Bruton's tyrosine kinase (BTK) for oncology indications. However, a formidable challenge for the pharmaceutical industry has been the identification of reversible, selective, potent molecules for inhibition of BTK. Herein, we report application of Tethering-fragment-based screens to identify low molecular weight fragments which were further optimized to improve on-target potency and ADME properties leading to the discovery of reversible, selective, potent BTK inhibitors suitable for pre-clinical proof-of-concept studies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Two sets of synthetic 21-23mer oligonucleotides with various types of 2'-position modifications have been studied with tandem mass spectrometry using ion trap collision-induced dissociation (IT-CID) and negative electron transfer (NET)-CID. A systematic study has been conducted to define the limitations of IT-CID in sequencing such 2'-chemically modified oligonucleotides. We found that IT-CID is sufficient in characterizing oligonucleotide sequences that do not contain DNA residues, where high sequence coverage can be achieved by performing IT-CID on multiple charge states. However, oligonucleotides containing DNA residues gave limited backbone fragmentation with IT-CID, largely due to dominant fragmentation at the DNA residue sites. To overcome this limitation, we employed the negative electron transfer to strip an electron from the multiply charged oligonucleotide anion. Then, the radical anion species formed in this reaction can fragment via an alternative radical-directed dissociation mechanism. Unlike IT-CID, NET-CID mainly generates a noncomplementary d/w ion series. Furthermore, we found that NET-CID did not show preferential dissociations at the DNA residue sites and thus generated higher sequence coverage for the studied oligonucleotide. Information from NET-CID of different charge states is not fully redundant such that the examination of multiple charge states can lead to more extensive sequence confirmation. This work demonstrates that the NET-CID is a valuable tool to provide high sequence coverage for chemically modified oligonucleotides, and such detailed characterization can serve as an important assay to control the quality of therapeutic oligonucleotides that are produced under the good manufacture practice (GMP) regulations.

We report the use of a fragment-based lead discovery method, Tethering with extenders, to discover a pyridinone fragment that binds in an adaptive site of the protein PDK1. With subsequent medicinal chemistry, this led to the discovery of a potent and highly selective inhibitor of PDK1, which binds in the 'DFG-out' conformation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.

We demonstrate a fragment-based lead discovery method that combines site-directed ligand discovery with dynamic combinatorial chemistry. Our technique targets dynamic combinatorial screening to a specified region of a protein by using reversible disulfide chemistry. We have used this technology to rapidly identify inhibitors of the drug target Aurora A that span the purine-binding site and the adaptive pocket of the kinase. The binding mode of a noncovalent inhibitor has been further characterized through crystallography.

We have identified a small-molecule inhibitor of tumor necrosis factor alpha (TNF-alpha) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-alpha activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-alpha subunits.

The fluorogenic reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABDF) attenuates the functional activity of the protein tyrosine phosphatase PTP1B by reacting selectively with a single cysteine residue, leaving other cysteines in the protein unmodified. This modification reduces Vmax without substantially affecting substrate binding (Km), indicative of an allosteric mode of inhibition. Consistent with this, the cysteine residue modified by ABDF, Cys 121, lies outside the catalytic site but makes interactions with residues that contact His 214, which has been shown to be important for catalysis. Cys 121 is highly conserved among phosphatases, and ABDF also inhibits TC-PTP and LAR. These findings illustrate that targeting cysteine residues outside catalytic sites may be exploited in allosterically regulating enzymes. Moreover, these results suggest a new strategy for inhibiting a promising diabetes target.

A technique has been developed to rapidly screen enzyme inhibitor candidates from complex mixtures, such as those created by combinatorial synthesis. Inhibitor libraries are screened by using immobilized enzyme technologies and electrospray ionization ion cyclotron resonance mass spectrometry. The library mixture is first sprayed into the mass spectrometer, and compounds are identified. The library is subsequently incubated with the immobilized enzyme of interest under the correct conditions (buffer, pH, temperature) by using an excess of enzyme to ensure a surplus of sites for ligand binding. The immobilized enzyme/inhibitor mixture is centrifuged, and an aliquot of supernatant is again analyzed by electrospray ionization mass spectrometry. Potential inhibitors are quickly identified by comparison of the spectra before and after incubation with the immobilized enzyme. Non-inhibitors show no change in ion intensity after incubation, whereas weak inhibitors exhibit a visible decrease in ion abundance. Once inhibitor candidates have been identified, the library is reinjected into the mass spectrometer, and tandem mass spectrometry is used to determine the structure of the inhibitor candidates as needed. This method has been successfully demonstrated by identifying inhibitors of the enzymes pepsin and glutathione S-transferase from a 19- and 17-component library, respectively. It is further shown that the immobilized enzyme can be recycled and reused for continuous screening of additional new libraries without adding additional enzyme.

Lipooligosaccharide (LOS) glycoforms from Haemophilus influenzae 2019 were profiled using the high-resolution and accurate mass capabilities of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Sequence and linkage for two previously unknown LOS glycoforms were subsequently obtained through MSn analyses on FT-ICR and quadrupole ion trap (qIT) instruments. MSn analysis of negative ion precursors confirmed structural details within the lipid moiety, while CID spectra of sodiated precursor ions provided monosaccharide sequence and linkage for the oligosaccharide portion of the molecule. Results obtained in this study indicate that extensive heterogeneity exists within the oligosaccharide moieties in LOS from H. influenzae 2019. More importantly, the data suggest that additional hexose moieties, which are added onto the LOS, are not simple extensions of one particular core structure but rather that structural isomers with different connectivities are present within the heterogeneous mixture.

A general oligosaccharide acid hydrolysis method, amenable to electrospray ionization mass spectrometry (ESI-MS), is described that allows for hydrolysis of glycosidic bonds for both hexose- and N-acetylhexosamine-containing oligosaccharides. The partial acid hydrolysis of oligosaccharides is obtained by using an acid-exchange resin as the acid catalyst. A ladder sequence of the glycan is produced in solution that is directly analyzed by ESI tandem mass spectrometry, employing both ion trap and Fourier transform ion cyclotron resonance mass spectrometers, to provide sequence and linkage information. Unlike traditional acid hydrolysis procedures, there is minimal degradation of monosaccharide residues or deacetylation of N-acetylhexosamines by employing this technique. It is further demonstrated that the stereochemistry of the released monosaccharides and the anomeric configuration within disaccharides is determined by direct derivatization of the hydrolysate with Zn(dien)-Cl2 followed by ESI-MS/MS.

The structure of oceanapiside, an antifungal alpha, omega-bis-aminohydroxylipid glycoside from the temperate marine sponge Oceanapia sp., was elucidated by a combination of 2D NMR, chemical degradation/correlation, and MALDI MS-MS spectrometry. Oceanapiside exhibits antifungal activity against Candida glabrata at 10 micrograms/mL (MIC).

A method is described to obtain multicollision dissociation threshold (MCDT) values. These values provide relative reaction thresholds for dissociation in the three major gas-phase fragmentation reactions of oligosaccharides complexed to alkali metal ions. The quasimolecular ions are produced using matrix-assisted laser desorption/ionization Fourier transform mass spectrometry. The MCDTs for alkali metal ion dissociation and glycosidic bond and cross-ring cleavages were resolved from the kinetic energy dependence of collision-induced dissociation (CID) products. The relative strengths of alkali metal ion binding to N,N'-diacetylchitobiose (chitobiose) and N,N',N"-triacetylchitotriose (chitotriose) were probed using sustained off-resonance irradiation (SORI) CID. Experiments to evaluate MCDT values and the method for obtaining them were performed by studying alkali metal ion coordinated crown ethers. Molecular dynamic simulations were also performed to provide insight into the alkali metal ion binding of chitin-based oligosaccharides. The relative dissociation thresholds of glycosidic bond cleavages and cross-ring cleavages were determined for various alkali metal ion coordinated oligosaccharides. The activation barriers of glycosidic bond cleavages were found to depend on the size of the alkali metal ion. Cross-ring cleavages were found to be independent of the alkali metal ion but dependent on linkage type. The results suggest that glycosidic bond cleavages are charge-induced while cross-ring cleavages are charge-remote processes.

A new anion dopant for oligosaccharides is developed for use in matrix-assisted laser desorption/ionization mass spectrometry. Two types of sulfate-attached quasimolecular ions are formed in the negative ion mode when neutral oligosaccharides are doped with dilute H2SO4 solutions. Under mild conditions, i.e., low H2SO4 concentration (approximately 10(-3) M) and threshold laser fluence, a sulfate adduct [M + HSO4]- is formed. With more concentrated H2SO4 solutions (approximately 10(-2) M) and higher laser fluence, in situ derivatization of the oligosaccharides occurs to produce an ion whose m/z corresponds to a sulfate derivative [M + HSO4 - H2O]-. Hydrogen sulfate appears to be a general anion dopant because it forms complexes with a wide variety of neutral oligosaccharides. Conversely, anionic oligosaccharides form neither the adduct nor the derivative. The combination of complex formation (with neutral oligosaccharides) and the deprotonation of acidic oligosaccharides allows simultaneous detection of the respective mixture.

A new technique for determining sequence and linkage information of underivatized oligosaccharides is developed using alkaline degradation and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). Alkaline degradation (also known as the "peeling" reaction) is a chemical degradation technique that only cleaves the glycosidic bond at the reducing end by beta-elimination to yield a new reducing end. The reaction products are sampled directly with minimal cleanup and monitored by MALDI-FTMS to elucidate the oligosaccharide sequence. Linkage information is provided by cross-ring cleavage fragmentation of the new reducing ends, created by either MALDI source fragmentation or sustained off-resonance irradiation collision-induced dissociation. This method is illustrated by the successful sequence and linkage determination of neutral, branched, fucosylated, and sialylated oligosaccharides. Experiments on differently linked disaccharides are also performed to determine the specificity of the cross-ring cleavage reactions. The power of this technique is enhanced by the Fourier transform mass analyzer, which provides high-resolution, exact mass, and facile tandem mass spectrometry experiments of MALDI-produced ions.

Collision-induced dissociation (CID) is used in an external source Fourier transform mass spectrometer (FTMS) equipped with matrix-assisted laser desorption/ionization (MALDI) to study a number of complex, branched oligosaccharides. The relative dissociation thresholds for various oligosaccharide fragmentation pathways have been calculated in terms of kinetic and center-of-mass frame energy. For two isomers of difucosyllacto-N-hexaose, the loss of the fucose sugar is always the lowest energy fragment observed and occurs at the same energy for both isomers when the oligosaccharide is coordinated to a sodium ion. When the oligosaccharide is complexed to cesium, the threshold for the removal of the fucose moiety increases, indicating that the cesium is involved in a coordination complex that stabilizes the sugar. MS/ MS/MS is performed on a sugar, mannose core, which does not readily fragment during MALDI. In all the sugars examined, CID produces additional structural information relative to MALDI/FTMS.

A design is presented involving two separate vacuum chambers to provide nearly simultaneous capabilities of liquid secondary ion mass spectrometry (LSIMS), matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI) in an external source Fourier transform mass spectrometer. The instrument consists of two vacuum chambers, one with five stages of differential pumping for a combined LSIMS/MALDI source. The chamber dedicated to ESI was formerly a three-stage chamber with LSIMS and electron ionization. Two additional stages were added with the ESI source. LSIMS and MALDI have similar vacuum requirements and were moved to a newly built chamber with two stages of pumping. We present our first results obtained on the new vacuum chamber. Data presented for the MALDI source show that, with only two stages of pumping, and with shorter radio frequency-only quadrupole rods for ion injection, spectra comparable to those obtained on the formerly three-stage instrument can be obtained. Characterization of the MALDI source and data on linear, cyclic, and branched oligosaccharides are given. Finally, the design of the secon chamber is proposed as a low-cost prototype for an external source FTMS instrument.