Estimating the Reliability of Low-Abundant Signals and Limited Replicate Measurements through MS2 Peak Area in SWATH.

Sequential windows acquisition of all theoretical fragment ions mass spectrometry (SWATH-MS) provides large-scale protein quantification with high accuracy and selectivity. Nevertheless, reliable quantification of low-abundant signals in complex samples remains challenging, as recently illustrated in a multicenter benchmark study of different label-free software tools. Here, the SWATH Replicates Analysis 2.0 template from Sciex is used to highlight that the relationship between the MS2 peak area and the variability can be described by a function. This functional relationship appears to be largely insensitive to variation in samples or acquisition conditions, suggesting a device-intrinsic property. By using a power regression, it is shown that the MS2 peak area can be used to predict the quantification repeatability without relying on replicate injections, thus contributing to high-throughput confident quantification of low-abundant signals with SWATH-MS.

Health crises due to infectious and communicable diseases : European preparedness and response tools in an international context.

The combination of changes in eating habits, ways of living, globalisation, extensive travelling and the migration of millions of people around the world may be contributing to increased health risks. Certainly, health crises today are proving highly complex. More and more people are travelling and may carry with them unexpected virus vectors such as mosquitoes. Preparedness is challenging and there is a need for action plans to safeguard the growing at-risk population. Health crises can potentially affect a large proportion of the population and may lead to a significant increase in mortality or to an abnormally high death rate. This should be integrated into the general concept of national and international surveillance in order to provide a prepared response in the event of crisis. This paper provides an inventory of the relevant laws, guidelines and tools in Europe (and to a lesser degree, beyond), and proposes answers to the health crisis problems associated with infectious and communicable diseases. In crisis management, communication is an important factor to consider. This paper can serve as a tool for people involved in crisis preparedness.

An application of mass spectrometry for quality control of biologicals: Highly sensitive profiling of plasma residuals in human plasma-derived immunoglobulin.

Thromboembolic events (TEE) associated to trace amounts of plasmatic activated coagulation factor XI (FXIa) in administrated immunoglobulin (Ig) have recently raised concerns and hence there is a need for highly sensitive profiling of residual plasma source proteins. This study aims to consider LC-ESI-QTOF data-dependent acquisition in combination with sample fractionation for this purpose. Sample fractionation proved mandatory to enable identification of plasma residuals. Two approaches were compared: Ig depletion with protein G - protein A affinity chromatography and low-abundant protein enrichment with a combinatorial peptide ligand library (ProteoMiner™, Bio-Rad). The latter allowed a higher number of identifications. Highly sensitive detection of prothrombotic FXIa was assessed with confident identification of a 1ng/mg spike. Moreover, different residuals compositions were profiled for various commercial Ig products. Using a quantitative label free analysis, a TEE-positive Ig batch was distinguished from other regular Ig products, with increased levels of FXIa but also other unique proteins. This could have prevented the recently observed TEE problems with Ig. The method is a convenient tool to better characterize Ig products after any plasma pool or manufacture process change, gaining insights in the product quality profile without any prior information required. BIOLOGICAL SIGNIFICANCE: This study characterized residual plasma proteins in Ig products, using bottom-up LC-MS/MS with conventional data-dependent acquisition, preceded by sample fractionation. Without any prior information or target-specific development, >30 proteins were identified in a commercial Ig product. Quality control relevance was demonstrated with the identification of FXIa spiked at 1ng/mg in Ig, which is below the minimal thrombotic dose of 3ng/mg observed in an in vivo model. Relative label-free quantitation highlighted significant differences in normalized abundances of residual proteins between Ig products. A TEE-positive batch was distinguished by unique profile of residual proteins, including FXIa but also various blood stream-regulator proteins (fibrinogen, angiotensinogen, antithrombin-III, complement component C8, …). Those results emphasize that MS screening is a relevant first-line test to prevent any undesired concentration of plasma impurities after a plasma pool or manufacturing process change.

A theoretical introduction to "combinatory SYBRGreen qPCR screening", a matrix-based approach for the detection of materials derived from genetically modified plants.

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. "Combinatory qPCR SYBRGreen screening" (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBRGREEN qPCR analysis based on four values: the C(t)- and T(m) values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, "Prime number tracing", matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBRGREEN qPCR methods and through application of a newly developed "prime number"-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product.

Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

Bringing scientists to the people--the Co-Extra website.

Disseminating and facilitating access to science-based information is a necessity to enable the public to make informed decisions about appropriate uses of biotechnology products. It is also one of the major objectives of Co-Extra, a European-funded project addressing co-existence of genetically modified organisms and non-genetically modified organisms in Europe and their traceability. To this end, a dynamic and interactive website has been developed as the core element of the Co-Extra external communication strategy. This website has been designed to make it attractive and accessible to a large audience in a very simple and practical manner, building on practical experiences gained in the development of other websites related to biotechnology and genetically modified organisms. The website delivers popularized information for the general public as well as scientific data meant primarily for the more expert readers. It also provides for various permanent tools allowing multidirectional interaction with its visitors. Content is displayed using a web-based platform, based on a sophisticated Content Management System. First results indicate a high level of interest from the general public and from experts, showing that the content of this website can contribute by communicating science-based information to improve awareness and understanding of biotechnology.