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More than half of all human proteins are glycosylated, making glycosylation one of the most abundant post-translational modifications in proteomics. N-Glycosylation is a prevalent and diverse type of glycosylation with key roles in regulating systems such as protein folding and host-pathogen recognition; without proper understanding of the heterogeneities of N-Glycosylation efforts to understand biological systems and efforts to combat the maladies that affect those systems will be hindered, knowingly and unknowingly. N-Glycosylation is a semi-stochastic process governed by local chemistries and enzymatic availability, and it is regulated by end process evaluation making modeling infeasible. This drives glycoproteomics to rely on observational data from tandem mass spectrometry; mass spectrometry is a powerful tool that comes with logistical and technical limitations on the availability and compatibility of data. N -Glycopeptides can be identified in tandem mass spectrometry data, but this is with greater uncertainty than traditional proteomics for a variety of factors. This uncertainty propagates into the quantification of these molecules, generating interdependent datasets with small sample sizes and high missing value rates. N-Glycans are inherently interrelated by the biosynthetic network that they’re processed in, and as a result they have a lot of shared information and chemical properties that make identification and quantification more difficult. While advances in N-Glycoproteomics continue there is still a lot needed for true and reliable understanding of quantitative N-Glycoproteomics. To make use of the existing data, an R-package called RAMZIS — Relative Assessment of m/z Identifications by Similarity — was developed. This toolkit focuses on data quality assessment and identifying broad differences between glycosylation sites. RAMZIS uses a series of permutation tests with a weighted Tanimoto similarity assessment, it provides researchers with information on their ability to use their data, the presence of outliers, the probable differentiability of glycosylation sites, and how to improve their future experimentations. Data Independent Acquisition (DIA) has enabled vast improvements in proteomic’s ability to quantify and identify proteins in complex samples, but these improvements cannot be directly applied to glycoproteomics. Glycoproteins are more heterogeneous than deglycosylated proteomic datasets and have lower overall signal, the latter compounding the issues made by the former. For glycoproteomics to make full use of the power of DIA and account for its idiosyncrasies, many bioinformatic advancements need to be made in glycopeptide identification, validation, and quantification. To this effort, we developed a python package called GlyLine as a framework to assess glycoproteomic DIA data; it tracks coeluting product ions of identified glycopeptides, splitting the signal from shared product ions to produce MS2 level quantifications of the identified glycopeptides and provide databases of information for further analysis. As glycoproteomics advances and comes into greater prominence, it is vital that experiments and bioinformatic workflows be repeatable, as quantitative glycoproteomic data are reported in many different ways that are often incompatible. We have worked with the MIRAGE Commission to develop a community based minimum reporting guideline for glycoproteomic experiments. / 2025-09-30T00:00:00Z

No / The Raman and mass spectra of 1,4-diphenylbuta-1,3-diene and several of its monomethyl and dimethyl homologues are reported and discussed, with a view to developing a spectroscopic protocol for detecting the presence and position of a methyl group in these compounds. Raman spectroscopy and mass spectrometry are shown to provide complementary information, by which the four available monomethyl homologues may be readily distinguished from each other and 1,4-diphenylbuta-1,3-diene itself. The utility of these 1,4-diarylbutadienes as model compounds for carotenoids and related materials, which may serve as indicators of extinct or extant extraterrestrial life, is considered.

No / No Abstract

Yes / The level of glutamine (Gln) deamidation in bone collagen provides information on the diagenetic history of bone but, in order to accurately assess the extent of Gln deamidation, it is important to minimise the conditions that may induce deamidation during the sample preparation. Here we report the results of a preliminary investigation of the variability in glutamine deamidation levels in an archaeological bone due to: a) sampling location within a bone; b) localised diagenesis; and c) sample preparation methods. We then investigate the effects of pre-treatment on three bone samples: one modern, one Medieval and one Pleistocene. The treatment of bone with acidic solutions was found to both induce deamidation and break down the collagen fibril structure. This is particularly evident in the Pleistocene material (~80,000 years BP) considered in this study. We show that ethylenediaminetetraacetic acid (EDTA), when used as an alternative to hydrochloric acid (HCl) demineralisation, induces minimal levels of deamidation and maintains the collagen fibril structure. Areas of bone exhibiting localised degradation are shown to be correlated with an increase in the levels of Gln deamidation. This indicates that the extent of Gln deamidation could provide a marker for diagenesis but that sampling is important, and that, whenever possible, subsamples should be taken from areas of the bone that are visually representative of the bone as a whole. Although validation of our observations will require analysis of a larger sample set, deamidation measurements could be a valuable screening tool to evaluate the suitability of bone for further destructive collagen analyses such as isotopic or DNA analysis, as well as assessing the overall preservation of bone material at a site. The measure of bone preservation may be useful to help conservators identify bones that may require special long-term storage conditions. / NERC (NE/J500197/1), Yorkshire Forward - Northern Way Initiative, Science City York, Gordon and Betty Moore Foundation, Leverhulme Trust

Capillary electrochromatography (CEC) is a rapidly developing liquid chromatographic technique in which electroosmotic flow (EOF) is used to propel mobile phase through the chromatographic column. The use of EOF results in reduced band dispersion when compared with pressurised flow, but narrow capillaries are required to avoid dispersion due to heating that arises from the required application of high electrical potentials. Measurement of UV absorbance in these narrow capillaries is therefore relatively insensitive, demanding improved detection methods. This work presents an alternative strategy that is based on the combination of ion trap accumulation with time-of-flight mass spectrometry. Electrospray is most often used to transfer analytes from solution to the gas phase, concomitant with ionisation, when interfacing CEC to mass spectrometry. The small volumetric flow rates encountered in CEC, however, raise the possibility of other types of interface being effective. The work presented here describes the development of a novel interface in which a pulsed IR laser is used to vaporise chromatographic eluent, followed by ionisation using a pulsed UV laser. Vaporisation and ionisation both occur within the ion trap to remove the possibility of transmission losses. Ionisation laser wavelength is varied to impart a degree of selectivity. The presence of vaporised solvent and analyte ions inside the trap offers the possibility of performing ion-molecule chemistry. In developing this instrument, the electrochromatographic column was separated from the interface by an electrically grounded junction and a transfer capillary. To preserve chromatographic efficiency, the fluid dynamics of this junction between the column and the transfer capillary were investigated both computationally and experimentally. Simulations of the fluid dynamics of the junction are presented. In order to test the interface without the intermittent, chromatographic, delivery of analyte, a continuous leak inlet was employed. The performance of the instrument was evaluated with polycyclic aromatic hydrocarbons because they are important environmental pollutants and because they are amenable to laser ionisation at 266 nm. Expressed as a number of theoretical plates per metre, an average chromatographic efficiency of 95,000 was obtained with a test mixture that consisted of acenaphthene, biphenyl, fluorene, naphthalene and phenanthrene. Furthermore, using the leak inlet, naphthalene was detected as a 100 nM solution in acetonitrile.

Imaging mass spectrometry (IMS) in microscope mode allows the spatially resolved molecular constitution of a large sample section to be analysed in a single experiment. If performed in a linear mass spectrometer, the applicability of microscope IMS is limited by a number of factors: the low mass resolving power of the employed ion optics; the time resolution afforded by the scintillator screen based particle detector and the multi-hit capability, per pixel, of the employed imaging sensor. To overcome these limitations, this thesis concerns the construction of an advanced ion optic employing a pulsed extraction method to gain a higher ToF resolution, the development of a bright scintillator screen with short emission lifetime, and the application of the Pixel Imaging Mass Spectrometry (PImMS) sensor with multi-mass imaging and time stamping capabilities. Initial experimental results employing a three electrode ion optic to spatially map ions emitted from a sample surface are presented. By applying a static electric potential a time-of-flight resolution of t/2Δt=54 and a spatial resolution of 20 μm are determined across a field-of-view of 4 mm diameter. While the moderate time-of-flight resolution only allows particles separated by a few Dalton to be distinguished, the instrument is used to demonstrate the multi-mass imaging capabilities of the PImMS sensor when being applied to image grid structures or tissue samples. An improved time-of-flight resolution is achieved by post extraction differential acceleration of a selected range of ions (up to 100 Da) using a newly developed five electrode ion optic. This modification is shown to correct the initial velocity spread of the ions coming off the sample surface, which yields an enhanced time-of-flight resolution of t/2Δt=2000 . The spatial resolution of the instrument is found to be 20 μm across a field-of-view of 4 mm. Adjusting the extraction field strength applied to the ion optic of the constructed mass spectrometer allows the optimised mass range to be tuned to any mass of interest. Ion images are recorded for various samples with comparable spatial and ToF resolution. Hence, studies on tissue sections and multi sample arrays become accessible with the improved design and operational principle of the microscope mode IMS instrument. A fast and efficient conversion of impinging ions into detectable flashes of light, which can consequently be recorded by a fast imaging sensor, is essential to maintain the achievable time-of-flight and spatial resolution of the IMS instrument constructed. In order to find a suitable fast and bright scintillator to be applied in a microchannel based particle detector, various inorganic and organic substances are characterised in terms of their emission properties following electron excitation. Poly-para-phenylene laser dye screens are found to show an outstanding performance among all substances analysed. An emission life time of below 4 ns and a brightness exceeding that of a P47 screen (industry standard) by a factor 2× is determined. No signal degradation is observed over an extended period, and the spatial resolution is found to be comparable to commercial imaging detectors. Hence, these scintillator screens are fully compatible with any ion imaging application requiring a high time resolution. In a further series of mass spectrometric experiments, ions are accelerated onto a scintillator mounted in front of a multi pixel photon counter. The charged particle impact stimulated the emission of a few photons, which are collected by the fast photon counter. Poly-para-phenylene laser dyes again show an outstanding efficiency for the conversion of ions into photons, resulting in a signal enhancement of up to 5× in comparison to previous experiments, which employed an inorganic LYSO scintillator.

The mass spectra of the compounds [Me 3 SiCH2 nSiCl 4 n (n=1-3) were studied in detail. MIKES and CID spectra were used in conjunction with the observance of metastable processes to develop consistent fragmentation schemes. Particular attention is drawn to the formation of charged and neutral species containing the silicon-carbon double bond, including 2-silaallene, under conditions of electron impact.

The existence of singly, doubly, and triply charged diatomic molecular ions was observed by using an Accelerator Mass Spectrometry (AMS) technique. The mean lifetimes of 3 MeV boron diatomic molecular ions were measured. No isotopic effects on the mean lifetimes of boron diatomic molecules were observed for charge state 3+. Also, the mean lifetime of SiF^3+ was measured.

Brevetoxins are natural neurotoxins that are produced by “red tide” algae. In this study, brevetoxin-1 and brevetoxin-2 were incubated with rat liver hepatocytes and rat liver microsomes, respectively. After clean-up steps, samples were analyzed by liquid chromatography-electrospray mass spectrometry (LC-ES-MS). Two metabolites were found for brevetoxin-1: brevetoxin-1-M1 (MW 900 Da), formed by converting one double bond in the E or F ring into a diol; and brevetoxin-1-M2 (MW 884 Da), a hydrolysis product of brevetoxin-1 involving opening of the lactone ring. The incubation study of brevetoxin-2 found two metabolites. Brevetoxin-2-M1 (MW 912 Da) was elucidated by negative mode LC-MS/MS to be the hydrolysis product of brevetoxin-2. The second metabolite (brevetoxin-2-M2, MW 896 Da) was deduced to be brevetoxin-3. All brevetoxins have high affinities for sodium ions. Attempts to obtain informative product ions from the collision induced decomposition (CID) of [M + Na]+ brevetoxin precursor ions only resulted in uninformative sodium ion signals. In our nano-electrospray experiments, the addition of ammonium fluoride resulted in the formation of the ammonium adduct or protonated brevetoxin with a concomitant decrease of the sodium adduct peak. Product ion spectra of [M + NH4]+ and [M + H]+ were similar and provided useful structural information. The optimal values for ammonium fluoride concentration and the cone voltage were experimentally determined. In negative mode electrospray, without additives, deprotonated molecules of brevetoxins do not appear in high abundances, and thus are not well-suited for CID experiments. Several anions were tested for their abilities to form brevetoxin-anion adducts by mixing ammonium salts of these anions with brevetoxin-2 and brevetoxin-3. Under CID, [M + Cl]-, [M + Br]-, [M + OAc]-, [M + HCOO]-, [M + NO3]- adducts all produced only the respective anions in CID experiments, and thus, gave no structural information. In contrast, upon CID, both [M + F]- and [M + HCO3]- precursor adducts gave structurally-informative fragment peaks that exhibited similarities to those of [M - H]- ions; the detailed fragmentation mechanisms are discussed. In comparison, fluoride is a better choice to study brevetoxins in negative ES-MS by the anionic adduct approach.