Abstract
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.
Highlights
In experiments simulating conditions for a potential chemoautotrophic origin of metabolism, simple inorganic C1 - or C2 -educts are used as precursors for the formation of organic molecules thatLife 2019, 9, 50; doi:10.3390/life9020050 www.mdpi.com/journal/lifeLife 2019, 9, 50 could have established the first “metabolic” reaction networks in the Universe [1,2]
The product mixtures obtained under these conditions are characterized by hundreds to thousands of compounds, most of them being at very low concentrations
This constraint is valid for many scenarios, which may be as different in their conceptual background as molecules formed on Earth under putative Hadean conditions or molecules formed in space and detected as product mixtures obtained from meteorites
Summary
In experiments simulating conditions for a potential chemoautotrophic origin of metabolism, simple inorganic C1 - or C2 -educts are used as precursors for the formation of organic molecules that. As a bottleneck of this method, the number of compounds that can be simultaneously identified is rather low [12] and both PC and TLC relied on the comparison with retention indices of reference standards and were error prone Some of these problems could be addressed by two-dimensional TLC, but more sophisticated techniques were necessary to characterize more products in these reaction mixtures. The technique is powerful in detecting reaction products from complex synthetic mixtures, such as compound cocktails obtained from “origin-of-life” experiments. Sub-ppm mass accuracy in conjunction with ultrahigh mass resolution allows for the direct annotation of the elemental composition for the detected ions over a high mass range up to m/z 700 (m/z 1000 in interpolations) [26] This annotation makes the characterization of even highly complex substance mixtures possible. To determine the growing complexity of this metabolic evolution, research of the “origin-of-life” benefitted from the evolution of analytical methods (at a different time scale) with ever more sophisticated procedures (Figure 1)
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