Advances in pre-processing and model generation for mass spectrometric data analysis

The analysis of complex signals as obtained by mass spectrometric measurements is complicated and needs an appropriate representation of the data. Thereby the kind of preprocessing, feature extraction as well as the used similarity measure are of particular importance. Focusing on biomarker analysis and taking the functional nature of the data into account this task is even more complicated. A new mass spectrometry tailored data preprocessing is shown, discussed and analyzed in a clinical proteom study compared to a standard setting

Three-dimensional interferometric, spectrometric, and planetary views of Procyon

We used a new realistic 3D radiative-hydrodynamical model atmosphere of Procyon generated with the Stagger Code and synthetic spectra computed with the radiative transfer code Optim3D to re-analyze interferometric and spectroscopic data from the optical to the infrared of Procyon. We compute intensity maps in two optical filters centered at 500 and 800 nm (MARK III) and one infrared filter centered at 2200 nm (VINCI). We constructed stellar disk images accounting for the center-to-limb variations and used them to derive visibility amplitudes and closure phases. We provide 3D limb-darkening coefficients in the optical as well as in the infrared. We show that visibility curves and closure phases show clear deviations from circular symmetry from the 3rd lobe on. These deviations are detectable with current interferometers using closure phases. We derive new angular diameters at different wavelengths with two independent methods based on 3D simulations. We find a diameter_Vinci = 5.390 \pm 0.03 mas that this is confirmed by an independent asteroseismic estimation. The resulting Teff is 6591 K, which is consistent with the infrared flux method determinations. We find also a value of the surface gravity log g = 4.01 \pm 0.03 that is larger by 0.05 dex from literature values. Spectrophotometric comparisons with observations provide very good agreement with the spectral energy distribution and photometric colors, allowing us to conclude that the thermal gradient of the simulation matches fairly well Procyon. Finally, we show that the granulation pattern of a planet hosting Procyon-like star has a non-negligible impact on the detection of hot Jupiters in the infrared using interferometry closure phases. It is then crucial to have a comprehensive knowledge of the host star to directly detect and characterize hot Jupiters. In this respect, RHD simulations are very important to reach this aim.Comment: Accepted for publication on Astronomy and Astrophysics, 14 pages, 12 figure

Advances in structure elucidation of small molecules using mass spectrometry

The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successive chapter discusses mass spectral fragmentation pathways, biotransformation reactions and drug metabolism studies, the mass spectral simulation and generation of in silico mass spectra, expert systems for mass spectral interpretation, and the use of computational chemistry to explain gas-phase phenomena. A single chapter discusses data handling for hyphenated approaches including mass spectral deconvolution for clean mass spectra, cheminformatics approaches and structure retention relationships, and retention index predictions for gas and liquid chromatography. The last section reviews the current state of electronic data sharing of mass spectra and discusses the importance of software development for the advancement of structure elucidation of small molecules

Structure analysis of biologically important prokaryotic glycopolymers

Of the many post-translational modifications organisms can undertake, glycosylation is the most prevalent and the most diverse. The research in this thesis focuses on the structural characterisation of glycosylation in two classes of glycopolymer (lipopolysaccharide (LPS) and glycoprotein) in two domains of life (bacteria and archaea). The common theme linking these subprojects is the development and application of high sensitivity analytical techniques, primarily mass spectrometry (MS), for studying prokaryotic glycosylation. Many prokaryotes produce glycan arrangements with extraordinary variety in composition and structure. A further challenge is posed by additional functionalities such as lipids whose characterisation is not always straightforward. Glycosylation in prokaryotes has a variety of different biological functions, including their important roles in the mediation of interactions between pathogens and hosts. Thus enhanced knowledge of bacterial glycosylation may be of therapeutic value, whilst a better understanding of archaeal protein glycosylation will provide further targets for industrial applications, as well as insight into this post- translational modification across evolution and protein processing under extreme conditions. The first sub-project focused on the S-layer glycoprotein of the halophilic archeaon Haloferax volcanii, which has been reported to be modified by both glycans and lipids. Glycoproteomic and associated MS technologies were employed to characterise the N- and O-linked glycosylation and to explore putative lipid modifications. Approximately 90% of the S-layer was mapped and N-glycans were identified at all the mapped consensus sites, decorated with a pentasaccharide consisting of two hexoses, two hexuronic acids and a methylated hexuronic acid. The O-glycans are homogeneously identified as a disaccharide consisting of galactose and glucose. Unexpectedly it was found that membrane-derived lipids were present in the S- layer samples despite extensive purification, calling into question the predicted presence of covalently linked lipid. The H. volcanii N-glycosylation is mediated by the products of the agl gene cluster and the functional characterisation of members of the agl gene cluster was investigated by MS analysis of agl-mutant strains of the S-layer. Burkholderia pseudomallei is the causative agent of melioidosis, a serious and often fatal disease in humans which is endemic in South-East Asia and other equatorial regions. Its LPS is vital for serum resistance and the O-antigen repeat structures are of interest as vaccine targets. B. pseudomallei is reported to produce several polysaccharides, amongst which the already characterised ‘typical’ O-antigen of K96243 represents 97% of the strains. The serologically distinct ‘atypical’ strain 576 produces a different LPS, whose characterisation is the subject of this research project. MS strategies coupled with various hydrolytic and chemical derivatisation methodologies were employed to define the composition and potential sequences of the O-antigen repeat unit. These MS strategies were complemented by a novel NMR technique involving embedding of the LPS into micelles. Taken together the MS and NMR data have revealed a highly unusual O-antigen structure for atypical LPS which is remarkably different from the typical O-antigen. The development of structural analysis tools in MS and NMR applicable to the illustrated types of glycosylation in these prokaryotes will give a more consistent approach to sugar characterisation and their modifications thus providing more informative results for pathogenicity and immunological studies as well as pathway comparisons.Open Acces

IMass time: The future, in future!

Joseph John Thomson discovered and proved the existence of electrons through a series of experiments. His work earned him a Nobel Prize in 1906 and initiated the era of mass spectrometry (MS). In the intervening time, other researchers have also been awarded the Nobel Prize for significant advances in MS technology. The development of soft ionization techniques was central to the application of MS to large biological molecules and led to an unprecedented interest in the study of biomolecules such as proteins (proteomics), metabolites (metabolomics), carbohydrates (glycomics), and lipids (lipidomics), allowing a better understanding of the molecular underpinnings of health and disease. The interest in large molecules drove improvements in MS resolution and now the challenge is in data deconvolution, intelligent exploitation of heterogeneous data, and interpretation, all of which can be ameliorated with a proposed IMass technology. We define IMass as a combination of MS and artificial intelligence, with each performing a specific role. IMass will offer advantages such as improving speed, sensitivity, and analyses of large data that are presently not possible with MS alone. In this study, we present an overview of the MS considering historical perspectives and applications, challenges, as well as insightful highlights of IMass

Optimization-Based Peptide Mass Fingerprinting for Protein Mixture Identification

*Motivation:* In current proteome research, peptide sequencing is probably the most widely used method for protein mixture identification. However, this peptide-centric method has its own disadvantages such as the immense volume of tandem Mass Spectrometry (MS) data for sequencing peptides. With the fast development of technology, it is possible to investigate other alternative techniques. Peptide Mass Fingerprinting (PMF) has been widely used to identify single purified proteins for more than 15 years. Unfortunately, this technique is less accurate than peptide sequencing method and cannot handle protein mixtures, which hampers the widespread use of PMF technique. If we can remove these limitations, PMF will become a useful tool in protein mixture identification. 
*Results:* We first formulate the problem of PMF protein mixture identification as an optimization problem. Then, we show that the use of some simple heuristics enables us to find good solutions. As a result, we obtain much better identification results than previous methods. Moreover, the result on real MS data can be comparable with that of the peptide sequencing method. Through a comprehensive simulation study, we identify a set of limiting factors that hinder the performance of PMF method in protein mixtures. We argue that it is feasible to remove these limitations and PMF can be a powerful tool in the analysis of protein mixtures

Advances in analytical tools and current statistical methods used in ultra-high-performance liquid chromatography-mass spectrometry of glycero-, glycerophospho- and sphingolipids

The review concentrates on the properties of analytical and statistical ultrahigh-performance liquid chromatographic (UHPLC) - mass spectrometric (MS) methods suitable for glycero-, glycerophospho- and sphingolipids in lipidomics published between the years 2017 2019. Trends and fluctuations of conventional and nano-UHPLC methods with MS and tandem MS detection were observed in context of analysis conditions and tools used for data-analysis. Whereas general workflow characteristics are agreed upon, more details related to the chromatographic methodology (i.e. stationary and mobile phase conditions) need evidently agreements. Lipid quantitation relies upon isotope-labelled standards in targeted analyses and fully standardless algorithm-based untargeted analyses. Furthermore, a wide spectrum of setups have shown potential for the elucidation of complex and large datasets by minimizing the risks of systematic misinterpretation like false positives. This kind of evaluation was shown to have increased importance and usage for cross-validation and data-analysis. (C) 2020 Elsevier B.V. All rights reserved.Peer reviewe

Identification of vascular endothelial growth factor receptor 3 (VEGFR3) as an in vitro and in vivo substrate of the Alzheimer's Disease linked protease BACE2

The protease ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) is a key drug target in Alzheimer’s disease (AD). It catalyzes the first step in the generation of the pathogenic amyloid ß (Aß) peptide and its inhibition is therefore a promising approach to prevent or delay the onset of AD. To date however, most inhibitory compounds do not discriminate between BACE1 and its close non-amyloidogenic homologue BACE2 and therefore may lead to undesired off target effects, resulting from BACE2 biology. Therefore, future compounds require a higher selectivity for BACE1 and a biomarker is required to confirm unimpaired in vivo BACE2 activity. To replace a long lasting depigmentation assay, which is the current standard for in vivo BACE2 activity monitoring, the blood plasma of BACE2 knockout mice (B2KO) was screened and the tyrosine kinase receptor Vascular Endothelial Growth Factor 3 (VEGFR3) was identified as a putative BACE2 substrate. Subsequently, VEGFR3 was thoroughly validated as an in vitro and in vivo BACE2 substrate and the BACE2 cleavage site was determined. In direct comparison to the pigmentation readout, plasma VEGFR3 performed superior and displayed higher sensitivity and lower variance. Importantly, reduction of VEGFR3 was also detectable in the plasma of BACE inhibitor treated non-human primates (NHP) and clinical trial participants, highlighting potential for applicability in the clinical context. To test whether BACE2 cleavage may be a novel mechanism to control VEGFR3 function, downstream events of VEGFR3 signaling were monitored in primary lymphatic endothelial cells (LECs). Impairment of BACE2 dependent VEGFR3 processing was accompanied by increased activation of the VEGFR3 dependent pathways AKT and ERK and resulted into enhanced transcription of the VEGFR3 inducible genes (FOXC2) and Delta-like 4 (DLL4). As a consequence, alterations in the morphological structure and drainage efficiency of lymphatic vessels and cannot be excluded in the periphery and central nervous system (CNS). Future developments in the BACE inhibitor field need to consider these implications and plasma VEGFR3 levels may be used to control for possible of target effects from BACE2 inhibition

Addressing the needs of traumatic brain injury with clinical proteomics.

BackgroundNeurotrauma or injuries to the central nervous system (CNS) are a serious public health problem worldwide. Approximately 75% of all traumatic brain injuries (TBIs) are concussions or other mild TBI (mTBI) forms. Evaluation of concussion injury today is limited to an assessment of behavioral symptoms, often with delay and subject to motivation. Hence, there is an urgent need for an accurate chemical measure in biofluids to serve as a diagnostic tool for invisible brain wounds, to monitor severe patient trajectories, and to predict survival chances. Although a number of neurotrauma marker candidates have been reported, the broad spectrum of TBI limits the significance of small cohort studies. Specificity and sensitivity issues compound the development of a conclusive diagnostic assay, especially for concussion patients. Thus, the neurotrauma field currently has no diagnostic biofluid test in clinical use.ContentWe discuss the challenges of discovering new and validating identified neurotrauma marker candidates using proteomics-based strategies, including targeting, selection strategies and the application of mass spectrometry (MS) technologies and their potential impact to the neurotrauma field.SummaryMany studies use TBI marker candidates based on literature reports, yet progress in genomics and proteomics have started to provide neurotrauma protein profiles. Choosing meaningful marker candidates from such 'long lists' is still pending, as only few can be taken through the process of preclinical verification and large scale translational validation. Quantitative mass spectrometry targeting specific molecules rather than random sampling of the whole proteome, e.g., multiple reaction monitoring (MRM), offers an efficient and effective means to multiplex the measurement of several candidates in patient samples, thereby omitting the need for antibodies prior to clinical assay design. Sample preparation challenges specific to TBI are addressed. A tailored selection strategy combined with a multiplex screening approach is helping to arrive at diagnostically suitable candidates for clinical assay development. A surrogate marker test will be instrumental for critical decisions of TBI patient care and protection of concussion victims from repeated exposures that could result in lasting neurological deficits