Open Search of Peptide Glycation Products from Tandem Mass Spectra.

Identification of chemically modified peptides in mass spectrometry (MS)-based glycation studies is a crucial yet challenging task. There is a need to establish a mode for matching tandem mass spectrometry (MS/MS) data, allowing for both known and unknown peptide glycation modifications. We present an open search approach that uses classic and modified peptide fragment ions. The latter are shifted by the mass delta of the modification. Both provide key structural information that can be used to assess the peptide core structure of the glycation product. We also leverage redundant neutral losses from the modification side chain, introducing a third ion class for matching referred to as characteristic fragment ions. We demonstrate that peptide glycation product MS/MS spectra contain multidimensional information and that most often, more than half of the spectral information is ignored if no attempt is made to use a multi-step matching algorithm. Compared to regular and/or modified peptide ion matching, our triple-ion strategy significantly increased the median interpretable fraction of the glycation product MS/MS spectra. For reference, we apply our approach for Amadori product characterization and identify all established diagnostic ions automatically. We further show how this method effectively applies the open search concept and allows for optimized elucidation of unknown structures by presenting two hitherto undescribed peptide glycation modifications with a delta mass of 102.0311 and 268.1768 Da. We characterize their fragmentation signature by integration with isotopically labeled glycation products, which provides high validity for non-targeted structure identification.

Nickel-organo compounds as potential enzyme precursors under simulated early Earth conditions.

The transition from inorganic catalysis through minerals to organic catalysis by enzymes is a necessary step in the emergence of life. Our work is elucidating likely reactions at the earliest moments of Life, prior to the existence of enzymatic catalysis, by exploring essential intersections between nickel bioinorganic chemistry and pterin biochemistry. We used a prebiotically-inspired acetylene-containing volcanic hydrothermal experimental environment to shed light on the efficient formation of nickel-organo complexes. The simplest bis(dithiolene)nickel complex (C2H2S2)2Ni was identified by UV/Vis spectroscopy, mass spectrometry, nuclear magnetic resonance. Its temporal progression and possible function in this simulated early Earth atmosphere were investigated by isolating the main bis(dithiolene)nickel species from the primordial experimental setup. Using this approach, we uncovered a significant diversity of nickel-organo compositions by identifying 156 elemental annotations. The formation of acetaldehyde through the subsequent degradation of these organo-metal complexes is intriguing, as it is reminiscent of the ability of Pelobacter acetylenicus to hydrate acetylene to acetaldehyde via its bis(dithiolene)-containing enzyme acetylene hydratase. As our findings mechanistically characterize the role of nickel sulfide in catalyzing the formation of acetaldehyde, this fundamental pre-metabolic reaction could play the role of a primitive enzyme precursor of the enzymatic acetylene metabolism and further strengthen the role of acetylene in the molecular origin of life.

Increased levels of a mycophenolic acid metabolite in patients with kidney failure negatively affect cardiomyocyte health.

Chronic kidney disease (CKD) significantly increases cardiovascular risk and mortality, and the accumulation of uremic toxins in the circulation upon kidney failure contributes to this increased risk. We thus performed a screening for potential novel mediators of reduced cardiovascular health starting from dialysate obtained after hemodialysis of patients with CKD. The dialysate was gradually fractionated to increased purity using orthogonal chromatography steps, with each fraction screened for a potential negative impact on the metabolic activity of cardiomyocytes using a high-throughput MTT-assay, until ultimately a highly purified fraction with strong effects on cardiomyocyte health was retained. Mass spectrometry and nuclear magnetic resonance identified the metabolite mycophenolic acid-ß-glucuronide (MPA-G) as a responsible substance. MPA-G is the main metabolite from the immunosuppressive agent MPA that is supplied in the form of mycophenolate mofetil (MMF) to patients in preparation for and after transplantation or for treatment of autoimmune and non-transplant kidney diseases. The adverse effect of MPA-G on cardiomyocytes was confirmed in vitro, reducing the overall metabolic activity and cellular respiration while increasing mitochondrial reactive oxygen species production in cardiomyocytes at concentrations detected in MMF-treated patients with failing kidney function. This study draws attention to the potential adverse effects of long-term high MMF dosing, specifically in patients with severely reduced kidney function already displaying a highly increased cardiovascular risk.

C2-addition patterns emerging from acetylene and nickel sulfide in simulated prebiotic hydrothermal conditions.

Chemical complexity is vital not only for the origin of life but also for biological evolution. The chemical evolution of a complex prebiotic mixture containing acetylene, carbon monoxide (CO), and nickel sulfide (NiS) has been analyzed with mass spectrometry as an untargeted approach to reaction monitoring. Here we show through isotopic 13C-labelling, multiple reaction products, encompassing diverse CHO and CHOS compounds within the complex reaction mixture. Molecules within the same chemical spaces displayed varying degrees of 13C-labelling, enabling more robust functional group characterization based on targeted investigations and differences in saturation levels among the described classes. A characteristic C2-addition pattern was detected in all compound classes in conjunction with a high diversity of thio acids, reminiscent of extant microbial C2-metabolism. The analysis involved a time-resolved molecular network, which unveiled the behavior of sulfur in the system. At the onset of the reaction, early formed compounds contain more sulfur atoms compared to later emerging compounds. These results give an essential insight into the still elusive role of sulfur dynamics in the origin of life. Moreover, our results provide temporally resolved evidence of the progressively increasing molecular complexity arising from a limited number of compounds.

Formation, stabilization and fate of acetaldehyde and higher aldehydes in an autonomously changing prebiotic system emerging from acetylene.

Many essential building blocks of life, including amino acids, sugars, and nucleosides, require aldehydes for prebiotic synthesis. Pathways for their formation under early earth conditions are therefore of great importance. We investigated the formation of aldehydes by an experimental simulation of primordial early earth conditions, in line with the metal-sulfur world theory in an acetylene-containing atmosphere. We describe a pH-driven, intrinsically autoregulatory environment that concentrates acetaldehyde and other higher molecular weight aldehydes. We demonstrate that acetaldehyde is rapidly formed from acetylene over a nickel sulfide catalyst in an aqueous solution, followed by sequential reactions progressively increasing the molecular diversity and complexity of the reaction mixture. Interestingly, through inherent pH changes, the evolution of this complex matrix leads to auto-stabilization of de novo synthesized aldehydes and alters the subsequent synthesis of relevant biomolecules rather than yielding uncontrolled polymerization products. Our results emphasize the impact of progressively generated compounds on the overall reaction conditions and strengthen the role of acetylene in forming essential building blocks that are fundamental for the emergence of terrestrial life.

Formation of vesicular structures from fatty acids formed under simulated volcanic hydrothermal conditions.

Microscopic compartmentalization is beneficial in synthetic chemistry and indispensable for the evolution of life to separate a reactive "inside" from a hydrolyzing "outside". Here, we show compartmentalization in aqueous solution containing mixtures of fatty acids up to 19 carbon atoms which were synthesized by one-pot reactions of acetylene and carbon monoxide in contact with nickel sulfide at 105 °C, reaction requirements which are compatible to Hadean Early Earth conditions. Based on confocal, dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, vesicle-like structures with diameters of 10-150 nm are formed after solvent extraction and resolubilisation. Moreover fluorescent dye was encapsulated into the structures proving their vesicular properties. This self-assembly could also have occurred on Early Earth as a crucial step in establishing simple membranes of proto-cells as a prerequisite in the evolution of metabolism and life.

Archeochemistry reveals the first steps into modern industrial brewing.

A historical beer, dated to the German Empire era, was recently found in northern Germany. Its chemical composition represents a unique source of insights into brewing culture of the late nineteenth century when pioneer innovations laid the foundations for industrial brewing. Complementary analytics including metabolomics, microbiological, sensory, and beer attribute analysis revealed its molecular profile and certify the unprecedented good storage condition even after 130 years in the bottle. Comparing its chemical signature to that of four hundred modern brews allowed to describe molecular fingerprints teaching us about technological aspects of historical beer brewing. Several critical production steps such as malting and germ treatment, wort preparation and fermentation, filtration and storage, and compliance with the Bavarian Purity Law left detectable molecular imprints. In addition, the aging process of the drinkable brew could be analyzed on a chemical level and resulted in an unseen diversity of hops- and Maillard-derived compounds. Using this archeochemical forensic approach, the historical production process of a culturally significant beverage could be traced and the ravages of time made visible.

Evolutionary Steps in the Analytics of Primordial Metabolic Evolution.

Experimental studies of primordial metabolic evolution are based on multi-component reactions which typically result in highly complex product mixtures. The detection and structural assignment of these products crucially depends on sensitive and selective analytical procedures. Progress in the instrumentation of these methods steadily lowered the detection limits to concentrations in the pico molar range. At the same time, conceptual improvements in chromatography, nuclear magnetic resonance (NMR) and mass spectrometry dramatically increased the resolution power as well as throughput, now, allowing the simultaneous detection and structural determination of hundreds to thousands of compounds in complex mixtures. In retrospective, the development of these analytical methods occurred stepwise in a kind of evolutionary process that is reminiscent of steps occurring in the evolution of metabolism under chemoautotrophic conditions. This can be nicely exemplified in the analytical procedures used in our own studies that are based on Wächtershäuser's theory for metabolic evolution under Fe/Ni-catalyzed volcanic aqueous conditions. At the onset of these studies, gas chromatography (GC) and GC-MS (mass spectrometry) was optimized to detect specific low molecular weight products (<200 Da) in a targeted approach, e.g., methyl thioacetate, amino acids, hydroxy acids, and closely related molecules. Liquid chromatography mass spectrometry (LC-MS) was utilized for the detection of larger molecules including peptides exceeding a molecular weight of 200 Da. Although being less sensitive than GC-MS or LC-MS, NMR spectroscopy benefitted the structural determination of relevant products, such as intermediates involved in a putative primordial peptide cycle. In future, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) seems to develop as a complementary method to analyze the compositional space of the products and reaction clusters in a non-targeted approach at unprecedented sensitivity and mass resolution (700,000 for m/z 250). Stable isotope labeling was important to differentiate between reaction products and artifacts but also to reveal the mechanisms of product formation. In this review; we summarize some of the developmental steps and key improvements in analytical procedures mainly used in own studies of metabolic evolution.