Paleo-diatom composition from Santa Barbara Basin deep-sea sediments: a comparison of 18S-V9 and diat-rbcL metabarcoding vs shotgun metagenomics

Sedimentary ancient DNA (sedaDNA) analyses are increasingly used to reconstruct marine ecosystems. The majority of marine sedaDNA studies use a metabarcoding approach (extraction and analysis of specific DNA fragments of a defined length), targeting short taxonomic marker genes. Promising examples are 18S-V9 rRNA (~121–130 base pairs, bp) and diat-rbcL (76 bp), targeting eukaryotes and diatoms, respectively. However, it remains unknown how 18S-V9 and diat-rbcL derived compositional profiles compare to metagenomic shotgun data, the preferred method for ancient DNA analyses as amplification biases are minimised. We extracted DNA from five Santa Barbara Basin sediment samples (up to ~11 000 years old) and applied both a metabarcoding (18S-V9 rRNA, diat-rbcL) and a metagenomic shotgun approach to (i) compare eukaryote, especially diatom, composition, and (ii) assess sequence length and database related biases. Eukaryote composition differed considerably between shotgun and metabarcoding data, which was related to differences in read lengths (~112 and ~161 bp, respectively), and overamplification of short reads in metabarcoding data. Diatom composition was influenced by reference bias that was exacerbated in metabarcoding data and characterised by increased representation of Chaetoceros, Thalassiosira and Pseudo-nitzschia. Our results are relevant to sedaDNA studies aiming to accurately characterise paleo-ecosystems from either metabarcoding or metagenomic data.

Data and Diversity Driven Development of a Shotgun Crystallisation Screen using the Protein Data Bank

Protein crystallisation has for decades been a critical and restrictive step in macromolecular structure determination via X-ray diffraction. Crystallisation typically involves a multi-stage exploration of the available chemical space, beginning with an initial sampling (screening) followed by iterative refinement (optimisation). Effective screening is important for reducing the number of optimisation rounds required, reducing the cost and time required to determine a structure. Here, we propose an initial screen (Shotgun II) derived from analysis of the up-to-date Protein Data Bank (PDB) and compare it with the previously derived (2014) Shotgun I screen. In an update to that analysis, we clarify that the Shotgun approach entails finding the crystallisation conditions which cover the most diverse space of proteins by sequence found in the PDB - which can be mapped to the well known Maximum Coverage problem in computer science. With this realisation we are able to apply a more effective algorithm for selecting conditions, such that the Shotgun II screen outperforms the Shotgun I screen both in protein coverage and quantity of data input. Our data demonstrates that the Shotgun I screen, compared with alternatives, has been remarkably successful over the seven years it has been in use, indicating that Shotgun II is likely to be a highly effective screen.

Does Filter Aided Sample Preparation (FASP) Provide Sufficient Method Linearity for Quantitative Plant Shotgun Proteomics?

Gel-free LC-based shotgun proteomics represents the current gold standard of proteome analysis due to its outstanding throughput, analytical resolution and reproducibility. Thereby, the efficiency of sample preparation, i.e., protein isolation, solubilization and proteolysis, directly affects the correctness and reliability of quantification, being therefore the bottle neck of shotgun proteomics. The desired performance of the sample preparation protocols can be achieved by application of detergents. However, these ultimately compromise reverse phase chromatographic separation and disrupt electrospray ionization. Filter aided sample preparation (FASP) represents an elegant approach to overcome these limitations. Although this method is comprehensively validated for cell proteomics, its applicability to plants and compatibility with plant-specific protein isolation protocols is still unknown, i.e., no data on linearity of underlying protein quantification methods for plant matrices is available. To fill this gap, we address here the potential of FASP in combination with two protein isolation protocols for quantitative analysis of pea (<i>Pisum sativum</i>) seed and <i>Arabidopsis thaliana</i> leaf proteomes by the shotgun approach. For this, in comprehensive spiking experiments with bovine serum albumin (BSA), we evaluated the linear dynamic range (LDR) of protein quantification in the presence of plant matrices. Further, we addressed the interference of two different plant matrices in quantitative experiments, accomplished with two alternative sample preparation workflows in comparison to conventional FASP-based digestion of cell lysates, considered here as a reference. Our results indicate very good applicability of FASP to quantitative plant proteomics with an only limited impact of the protein isolation technique on the methods overall performance.<br>

Does Filter Aided Sample Preparation (FASP) Provide Sufficient Method Linearity for Quantitative Plant Shotgun Proteomics?

Gel-free LC-based shotgun proteomics represents the current gold standard of proteome analysis due to its outstanding throughput, analytical resolution and reproducibility. Thereby, the efficiency of sample preparation, i.e., protein isolation, solubilization and proteolysis, directly affects the correctness and reliability of quantification, being therefore the bottle neck of shotgun proteomics. The desired performance of the sample preparation protocols can be achieved by application of detergents. However, these ultimately compromise reverse phase chromatographic separation and disrupt electrospray ionization. Filter aided sample preparation (FASP) represents an elegant approach to overcome these limitations. Although this method is comprehensively validated for cell proteomics, its applicability to plants and compatibility with plant-specific protein isolation protocols is still unknown, i.e., no data on linearity of underlying protein quantification methods for plant matrices is available. To fill this gap, we address here the potential of FASP in combination with two protein isolation protocols for quantitative analysis of pea (<i>Pisum sativum</i>) seed and <i>Arabidopsis thaliana</i> leaf proteomes by the shotgun approach. For this, in comprehensive spiking experiments with bovine serum albumin (BSA), we evaluated the linear dynamic range (LDR) of protein quantification in the presence of plant matrices. Further, we addressed the interference of two different plant matrices in quantitative experiments, accomplished with two alternative sample preparation workflows in comparison to conventional FASP-based digestion of cell lysates, considered here as a reference. Our results indicate very good applicability of FASP to quantitative plant proteomics with an only limited impact of the protein isolation technique on the methods overall performance.<br>

Quantitative Label-Free Comparison of the Metabolic Protein Fraction in Old and Modern Italian Wheat Genotypes by a Shotgun Approach

Wheat represents one of the most important cereals for mankind. However, since wheat proteins are also the causative agent of several adverse reactions, during the last decades, consumers have shown an increasing interest in the old wheat genotypes, which are generally perceived as more “natural” and healthier than the modern ones. Comparison of nutritional value for modern and old wheat genotypes is still controversial, and to evaluate the real impact of these foods on human health comparative experiments involving old and modern genotypes are desirable. The nutritional quality of grain is correlated with its proteomic composition that depends on the interplay between the genetic characteristics of the plant and external factors related to the environment. We report here the label-free shotgun quantitative comparison of the metabolic protein fractions of two old Sicilian landraces (Russello and Timilia) and the modern variety Simeto, from the 2010–2011 and 2011–2012 growing seasons. The overall results show that Timilia presents the major differences with respect to the other two genotypes investigated. These differences may be related to different defense mechanisms and some other peculiar properties of these genotypes. On the other hand, our results confirm previous results leading to the conclusion that with respect to a nutritional value evaluation, there is a substantial equivalence between old and modern wheat genotypes. Data are available via ProteomeXchange with identifier <PXD024204>.

Grape Seeds Proanthocyanidins: Advanced Technological Preparation and Analytical Characterization

A “green” solvent-free industrial process (patent pending) is here described for a grape seed extract (GSE) preparation (Ecovitis™) obtained from selected seeds of Veneto region wineries, in the northeast of Italy, by water and selective tangential flow filtration at different porosity. Since a comprehensive, non-ambiguous characterization of GSE is still a difficult task, we resorted to using an integrated combination of gel permeation chromatography (GPC) and electrospray ionization high resolution mass spectrometry (ESI-HRMS). By calibration of retention time and spectroscopic quantification of catechin as chromophore, we succeeded in quantifying GPC polymers up to traces at n = 30. The MS analysis carried out by the ESI-HRMS method by direct-infusion allows the detection of more than 70 species, at different polymerization and galloylation, up to n = 13. This sensitivity took advantage of the nanoscale shotgun approach, although paying the limit of missed separation of stereoisomers. GPC and MS approaches were remarkably well cross-validated by overlapping results. This simple integrated analytical approach has been used for quality control of the production of Ecovitis™. The emerging feature of Ecovitis™ vs. a popular benchmark in the market, produced by a different technology, is the much lower content of species at low n and the corresponding increase of species at high n.

The development of mature gait patterns in children during walking and running

Purpose We sought to identify the developing maturity of walking and running in young children. We assessed gait patterns for the presence of flight and double support phases complemented by mechanical energetics. The corresponding classification outcomes were contrasted via a shotgun approach involving several potentially informative gait characteristics. A subsequent clustering turned out very effective to classify the degree of gait maturity. Methods Participants (22 typically developing children aged 2–9 years and 7 young, healthy adults) walked/ran on a treadmill at comfortable speeds. We determined double support and flight phases and the relationship between potential and kinetic energy oscillations of the center-of-mass. Based on the literature, we further incorporated a total of 93 gait characteristics (including the above-mentioned ones) and employed multivariate statistics comprising principal component analysis for data compression and hierarchical clustering for classification. Results While the ability to run including a flight phase increased with age, the flight phase did not reach 20% of the gait cycle. It seems that children use a walk-run-strategy when learning to run. Yet, the correlation strength between potential and kinetic energies saturated and so did the amount of recovered mechanical energy. Clustering the set of gait characteristics allowed for classifying gait in more detail. This defines a metric for maturity in terms of deviations from adult gait, which disagrees with chronological age. Conclusions The degree of gait maturity estimated statistically using various gait characteristics does not always relate directly to the chronological age of the child.