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

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 (ProteoMinerTm, Bio-Rad). The latter allowed a higher number of identifications. Highly sensitive detection of prothrombotic FXIa was assessed with confident identification of a 1 ng/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 1 ng/mg in Ig, which is below the minimal thrombotic dose of 3 ng/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

Animal cell cultures: Risk Assessment and biosafety recommendations

During the last three decades, animal cell culturing hasbeen essential for biomedical research and biotechnologicalactivities in general. Along with this increasing importance,biosafety concerns have pointed to the risks of manipulatinganimal cell cultures for human health and the environment. Amaximal reduction of these risks necessitates a thorough riskassessment of the cell cultures used. It involves an evaluationof both the intrinsic properties of the cell culture, includingsubsequent properties acquired as a result of genetic modification,and the possibility that the cell culture may inadvertentlyor deliberately become contaminated with pathogens.The latter is a major hazard associated with the manipulationof animal cell cultures, as adventitious agents may be pathogenicand have a better capacity to survive in unfavorableconditions. Consequently, most of the containment measuresprimarily aim at protecting cells from adventitious contamination.Therefore, a comprehensive evaluation of the risks encounteredduring the handling of cell cultures should includeconsiderations regarding the type of manipulation as well. Asa rule, cell cultures known to harbor an infectious etiologicagent should be manipulated in compliance with containmentmeasures recommended for the etiologic agent. With the exceptionof very well-characterized cell cultures for which theuse of a type II biosafety cabinet depends on the origin of thecells, work with cell cultures from human or primate originshould generally and minimally be performed under containmentlevel 2 using a type II biosafety cabinet. In every case,containment measures should minimize adventitious contaminationof the cell cultures and offer a maximal protection ofhuman health and the environment.</p

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 (ProteoMinerTm, Bio-Rad). The latter allowed a higher number of identifications. Highly sensitive detection of prothrombotic FXIa was assessed with confident identification of a 1 ng/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 1 ng/mg in Ig, which is below the minimal thrombotic dose of 3 ng/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