Industrial-scale proteomics: from liters of plasma to chemically synthesized proteins

Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.     

In vitro and in silico processes to identify differentially expressed proteins

We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g. by taking advantage of the powerful laboratory information management system we developed. The differential capacity of our platform is validated by detecting known markers in a real sample and by a spiking experiment. We introduce an innovative two-dimensional (2-D) plot for displaying identification results combined with chromatographic data. This 2-D plot is very convenient for detecting differential proteins. We also adapt standard multivariate statistical techniques to show that peptide identification scores can be used for reliable and sensitive differential studies. The interest of the protein separation approach we generally apply is justified by numerous statistics, complemented by a comparison with a simple shotgun analysis performed on a small volume sample. By introducing an automatic integration step after mass spectrometry data identification, we are able to search numerous databases systematically, including the human genome and expressed sequence tags. Finally, we explain how rigorous data processing can be combined with the work of human experts to set high quality standards, and hence obtain reliable (false positive < 0.35%) and nonredundant protein identifications.     

Isoenzymatic strains of Trypanosoma cruzi: recent or ancient, homogeneous or heterogeneous origin?

Genetic variability studied among 13 Trypanosoma cruzi laboratory reference stocks confirms the existence of three principal isozymic categories. As the main part of enzymic variability is now recognized, it seems possible to attribute any stock to the taxon T. cruzi with safety. Genetic parameters allow to infer two main hypotheses relative to the evolutionary origin of these categories: ancient origin (either by mitotic evolution or by speciation), or recent origin (by clone sampling among a sexual ancestral population). The possibility of a heterogeneous origin of the strains is discussed.     

Ecosystem engineering in the Quaternary of the West Coast of South Africa

Despite advances in our understanding of the geographic and temporal scope of the Paleolithic record, we know remarkably little about the evolutionary and ecological consequences of changes in human behavior. Recent inquiries suggest that human evolution reflects a long history of interconnections between the behavior of humans and their surrounding ecosystems (e.g., niche construction). Developing expectations to identify such phenomena is remarkably difficult because it requires understanding the multi‐generational impacts of changes in behavior. These long‐term dynamics require insights into the emergent phenomena that alter selective pressures over longer time periods which are not possible to observe, and are also not intuitive based on observations derived from ethnographic time scales. Generative models show promise for probing these potentially unexpected consequences of human‐environment interaction. Changes in the uses of landscapes may have long term implications for the environments that hominins occupied. We explore other potential proxies of behavior and examine how modeling may provide expectations for a variety of phenomena.     

Histidine 416 of the periplasmic binding protein NikA is essential for nickel uptake in Escherichia coli

Escherichia coli require nickel for the synthesis of [NiFe] hydrogenases under anaerobic growth conditions. Nickel import depends on the specific ABC-transporter NikABCDE encoded by the nik operon, which deletion causes the complete abolition of hydrogenase activity. We have previously postulated that the periplasmic binding protein NikA binds a natural metallophore containing three carboxylate functions that coordinate a Ni(II) ion, the fourth ligand being His416, the only direct metal-protein contact, completing a square-planar coordination for the metal. The crystal structure of the H416I mutant showed no electron density corresponding to a metal-chelator complex. In vivo experiments indicate that the mutation causes a significant decrease in nickel uptake and hydrogenase activity. These results confirm the essential role of His416 in nickel transport by NikA.     

Mobilisation of nutrients and transport via the xylem sap in a shrub (Ligustrum ovalifolium) during spring growth: N and C compounds and interactions

In open-field soilless culture there can be great deal of leaching, particularly in rainy springs. Ligneous plants have the capacity to store large quantities of nutrients in perennial organs. Knowledge of the plant's internal nutrient mobilisation during spring to supply growing organs could lead to reduction of fertiliser application. To quantify the fraction of storage mobilisation available for growth of new organs during spring, Ligustrum ovalifolium shrubs were grown for 2 years with or without fertilisation in the second spring. Nitrogen (N) absorption and N and carbon (C) mobilisation from storage were followed during spring growth via the sap quality. A mathematical combination of the sap composition with flow velocity provided the transported quantities of N and C. Nitrogen and C mobilisation towards new shoots took place during all the spring growth from bud break onwards. In unfertilised plants, C was mobilised primarily as sugars (stachyose, mannose and sucrose) and starch. In fertilised plants, the same sugars were transported in the xylem sap, but at lower concentrations. Stachyose concentration was lower in fertilised than in unfertilised plants and decreased during spring growth. Nitrogen was transported in the xylem sap mainly as amino acids in both fertilisation treatments. Glutamine was the predominant form at bud break and during shoot elongation. In fertilised plants, arginine became predominant after shoot elongation, and was related to low C availability. The interactions of N with C are discussed; specifically, insufficient availability of N limits the use of C, more of which is directed to aerial organs by sap flow.     

Applications of a microplate reader in yeast physiology research

Microplate readers have been useful assistants of researchers for several decades. This work is focused on the applications of a simple absorbance microplate reader in yeast physiology research, and its advantages and limitations in comparison with alternative methods are discussed. The two main procedures involved are measuring growth curves and monitoring the pH changes of medium using two different pH indicators. We suggest mathematical formulas for converting absorbance data into pH values. With a microplate reader as many as 96 samples can be simultaneously analyzed, while medium consumption is minimized to 100 microL per sample. The results can be observed in 24-48 h (for growth curves) or in 1-3 h (for pH changes) with minimal hands-on time required.