Here, we present liquid extraction surface analysis (LESA) coupled with electron-induced dissociation (EID) mass spectrometry in a Fourier-transform ion cyclotron resonance mass spectrometer for the analysis of small organic pharmaceutical compounds directly from dosed tissue. First, the direct infusion electrospray ionisation EID and collision-induced dissociation (CID) behaviour of erlotinib, moxifloxacin, clozapine and olanzapine standards were compared. EID mass spectra were also compared with experimental or reference electron impact ionisation mass spectra. The results show that (with the exception of erlotinib) EID and CID result in complementary fragment ions. Subsequently, we performed LESA EID MS/MS and LESA CID MS/MS on singly charged ions of moxifloxacin and erlotinib extracted from a thin tissue section of rat kidney from a cassette-dosed animal. Both techniques provided structural information, with the majority of peaks observed for the drug standards also observed for the tissue-extracted species. Overall, these results demonstrate the feasibility of LESA EID MS/MS of drug compounds from dosed tissue and extend the number of molecular structures for which EID behaviour has been determined.
A modified Kendrick Mass Defect (KMD) analysis was applied to the analysis of polycyclic aromatic hydrocarbons (PAHs) and fullerenes in the diffusion flame from a handheld butane torch.
A collision induced dissociation (CID) structure for lossless ion manipulations (SLIM) module is introduced and coupled to a quadrupole time-of-flight (QTOF) mass spectrometer. The SLIM CID module was mounted after an ion mobility (IM) drift tube to enable IM/CID/MS studies. The efficiency of CID was studied by using the model peptide leucine enkephalin. CID efficiencies (62%) compared favorably with other beam-type CID methods. Additionally, the SLIM CID module was used to fragment a mixture of nine peptides after IM separation. This work also represents the first application of SLIM in the 0.3 to 0.5 Torr pressure regime, an order of magnitude lower in pressure than previously studied.
Proof of concept evidence is presented for a new method for the determination of isoaspartate, an important post-translational modification. Chemical derivatization is performed using common reagents for the modification of carboxylic acids and shown to yield suitable diagnostic information with regard to isomerization at the aspartate residue. The diagnostic gas phase chemistry is probed by collision-induced dissociation mass spectrometry, on the timescale of the MS experiment and semi-quantitative calibration of the percentage of isoaspartate in a peptide sample is demonstrated.
The analysis of many hydrogen exchange (HX) experiments depends on knowledge of exchange rates expected for the unstructured protein under the same conditions. We present here some minor adjustments to previously calibrated values and a stringent test of their accuracy.
Ion mobility mass spectrometry (IM-MS) holds great potential for structural glycobiology, in particular in its ability to resolve glycan isomers. Generally, IM-MS has largely been applied to intact glycoconjugate ions with reports focusing on the separation of different adduct types. Here, we explore IM separation and report the collision cross section (CCS) of complex type N-glycans and their fragments in negative ion mode following collision-induced dissociation (CID). CCSs of isomeric fragment ions were found, in some cases, to reveal structural details that were not present in CID spectra themselves. Many fragment ions were confirmed as possessing multiple structure, details of which could be obtained by comparing their drift time profiles to different glycans. By using fragmentation both before and after mobility separation, information was gathered on the fragmentation pathways producing some of the ions. These results help demonstrate the utility of IM and will contribute to the growing use of IM-MS for glycomics.
The development of tandem ion mobility spectroscopy (IMS) known as IMS-IMS has led to extensive research into isomerizations of isolated molecules. Many recent works have focused on the retinal chromophore which is the optical switch used in animal vision. Here, we study a shortened derivative of the chromophore, which exhibits a rich IM spectrum allowing for a detailed analysis of its isomerization pathways, and show that the longer the chromophore is, the lower the barrier energies for isomerization are.
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.
We report distinctive spectroscopic fingerprints of the monosaccharide standards GalNAc4S and GalNAc6S by coupling mass spectrometry and ion spectroscopy in the 3-μm range. The disaccharide standards CSA and CSC are used to demonstrate the applicability of a novel approach for the analysis of sulfate position in GalNAc-containing glycosaminoglycans. This approach was then used for the analysis of a sample containing CSA and CSC disaccharides. Finally, we discuss the generalization of the coupling of mass spectrometry with ion spectroscopy for the structural analysis of glycosaminoglycans on a tetrasaccharide from dermatan sulfate source.
Hydrogen/deuterium exchange coupled with high-resolution mass spectrometry was successfully applied for the identification of A-type tetrameric, pentameric, and hexameric procyanidins in peanut skin. This extended a previous study on isomeric cyclic B-type unconventional tetramer, pentamer, and hexamer procyanidins found in wine and cranberries. Not only had the method successfully identified the procyanidins with a single A-linkage (e.g., tetrameric m/z 1153.2608) by means of distinguishing them from their isomeric cyclic B-type analogues, but this method also worked for procyanidins with two or more A-linkages (such as the tetrameric m/z 1151.2452). As a further consequence, B-type cyclic pentamers and hexamers in wine have been elucidated with hydrogen/deuterium exchange (HDX) for the first time.
Mixtures of pollen grains of three different species (Corylus avellana, Alnus cordata, and Pinus sylvestris) were investigated by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF imaging MS). The amount of pollen grains was reduced stepwise from >?10 to single pollen grains. For sample pretreatment, we modified a previously applied approach, where any additional extraction steps were omitted. Our results show that characteristic pollen MALDI mass spectra can be obtained from a single pollen grain, which is the prerequisite for a reliable pollen classification in practical applications. MALDI imaging of laterally resolved pollen grains provides additional information by reducing the complexity of the MS spectra of mixtures, where frequently peak discrimination is observed. Combined with multivariate statistical analyses, such as principal component analysis (PCA), our approach offers the chance for a fast and reliable identification of individual pollen grains by mass spectrometry.
Analyzing mass spectrometry imaging data can be laborious and time consuming, and as the size and complexity of datasets grow, so does the need for robust automated processing methods. We here present a method for comprehensive, semi-targeted discovery of molecular distributions of interest from mass spectrometry imaging data, using widely available image similarity scoring algorithms to rank images by spatial correlation. A fast and powerful batch search method using a MATLAB implementation of structural similarity (SSIM) index scoring with a pre-selected reference distribution is demonstrated for two sample imaging datasets, a plant metabolite study using Artemisia annua leaf, and a drug distribution study using maraviroc-dosed macaque tissue.
Structural characterization of intrinsically disordered proteins (IDPs) has been a major challenge in the field of protein science due to limited capabilities to obtain full-length high-resolution structures. Native ESI-MS with top-down MS was utilized to obtain structural features of protein-ligand binding for the Parkinson’s disease-related protein, α-synuclein (αSyn), which is natively unstructured. Binding of heavy metals has been implicated in the accelerated formation of αSyn aggregation. Using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, native top-down MS with various fragmentation methods, including electron capture dissociation (ECD), collisional activated dissociation (CAD), and multistage tandem MS (MS3), deduced the binding sites of cobalt and manganese to the C-terminal region of the protein. Ion mobility MS (IM-MS) revealed a collapse toward compacted states of αSyn upon metal binding. The combination of native top-down MS and IM-MS provides structural information of protein-ligand interactions for intrinsically disordered proteins.
Oligosaccharides have diverse functions in biological systems. However, the structural determination of oligosaccharides remains difficult and has created a bottleneck in carbohydrate research. In this study, a new approach for the de novo structural determination of underivatized oligosaccharides is demonstrated. A low-energy collision-induced dissociation (CID) of sodium ion adducts was used to facilitate the cleavage of desired chemical bonds during the dissociation. The selection of fragments for the subsequent CID was guided using a procedure that we built from the understanding of the saccharide dissociation mechanism. The linkages, anomeric configurations, and branch locations of oligosaccharides were determined by comparing the CID spectra of oligosaccharide with the fragmentation patterns based on the dissociation mechanism and our specially prepared disaccharide CID spectrum database. The usefulness of this method was demonstrated to determine the structures of several mannose trisaccharides. This method can also be applied in the structural determination of oligosaccharides larger than trisaccharides and containing hexose other than mannose if authentic standards are available.
Coaxial electrospray has been used effectively for several dual-emitter applications, but has not been utilized for the study of rapid in-source chemistry. In this paper, we report the fabrication of a coaxial, micro-volume dual-emitter through the modification of a manufacturer’s standard electrospray probe. This modification creates rapid mixing inside the Taylor cone and the ability to manipulate fast reactions using a variety of solvents and analytes. We demonstrate its potential as a low-cost, dual-emitter assembly for diverse applications through three examples: relative ionization in a biphasic electrospray, hydrogen-deuterium exchange, and protein supercharging.
A method to facilitate the characterization of stapled or cyclic peptides is reported via an arginine-selective derivatization strategy coupled with MS/MS analysis. Arginine residues are converted to ornithine residues through a deguanidination reaction that installs a highly selectively cleavable site in peptides. Upon activation by CID or UVPD, the ornithine residue cyclizes to promote cleavage of the adjacent amide bond. This Arg-specific process offers a unique strategy for site-selective ring opening of stapled and cyclic peptides. Upon activation of each derivatized peptide, site-specific backbone cleavage at the ornithine residue results in two complementary products: the lactam ring-containing portion of the peptide and the amine-containing portion. The deguanidination process not only provides a specific marker site that initiates fragmentation of the peptide but also offers a means to unlock the staple and differentiate isobaric stapled peptides.
There is considerable potential for the use of ion mobility mass spectrometry in structural glycobiology due in large part to the gas-phase separation attributes not typically observed by orthogonal methods. Here, we evaluate the capability of traveling wave ion mobility combined with negative ion collision-induced dissociation to provide structural information on N-linked glycans containing multiple fucose residues forming the Lewisx and Lewisy epitopes. These epitopes are involved in processes such as cell-cell recognition and are important as cancer biomarkers. Specific information that could be obtained from the intact N-glycans by negative ion CID included the general topology of the glycan such as the presence or absence of a bisecting GlcNAc residue and the branching pattern of the triantennary glycans. Information on the location of the fucose residues was also readily obtainable from ions specific to each antenna. Some isobaric fragment ions produced prior to ion mobility could subsequently be separated and, in some cases, provided additional valuable structural information that was missing from the CID spectra alone.
We report on the performance of a cryogenic 2D linear ion trap (cryoLIT) that is shown to be mass-selective in the temperature range of 17–295 K. As the cryoLIT is cooled, the ejection voltages during the mass instability scan decrease, which results in an effective mass shift to lower m/z relative to room temperature. This is attributed to a decrease in trap radius caused by thermal contraction. Additionally, the cryoLIT generates reproducible mass spectra from day-to-day, and is capable of performing stored waveform inverse Fourier transform (SWIFT) mass isolation of fragile N2-tagged ions for the purpose of background-free infrared dissociation spectroscopy.
Hydrogen deuterium exchange measured by mass spectrometry (HDX-MS) is a commonly used technique for studying the structural dynamics of proteins in solution. The first part of any bottom-up HDX-MS experiment is to identify the peptides generated from a digestion step. This requires manual inspection of the identified peptides to determine their use for HDX-MS analysis, which is a time-consuming task. Throughout the literature, there have been different approaches for removing peptides that do not yield quantifiable HDX information. This includes using validity scores from the software used in the generation of the peptide map and that the peptide should be found in two out of three technical replicate experiments. Here, we analyze the previously available methods for filtering the identified peptides in regard to their ability to predict whether a peptide will provide quantifiable HDX-MS data or not. We also present a new score-based system relying on a combination of MS/MS parameters that offers an improved method for separating quantifiable peptides from the nonquantifiable. Using this score-based method reduces the number of peptide spectra that needs to be manually inspected and thereby the time spent curating HDX-MS data.
The structural study of glycans and glycoconjugates is essential to assign their roles in homeostasis, health, and disease. Once dominated by nuclear magnetic resonance spectroscopy, mass spectrometric methods have become the preferred toolbox for the determination of glycan structures at high sensitivity. The patterns of such structures in different cellular states now allow us to interpret the sugar codes in health and disease, based on structure-function relationships. Dr. Catherine E. Costello was the 2017 recipient of the American Society for Mass Spectrometry’s Distinguished Contribution Award. In this Perspective article, we describe her seminal work in a historical and geographical context and review the impact of her research accomplishments in the field.
