An innovative standard for LC-MS-based HCP profiling and accurate quantity assessment: Application to batch consistency in viral vaccine samples.

Biotherapeutics, molecules produced from biological systems, require rigorous purification steps to remove impurities including host cell proteins (HCPs). Regulatory guidelines require manufacturers to monitor process-related impurities along the purification workflow. Mass spectrometry (MS) has recently been considered as a complementary method to the well-established ELISA for HCPs quantification, since it has the advantage of unambiguously identifying individual HCP. In this study, we developed an innovative standard dedicated to MS-based HCP profiling analysis in order to monitor the consistency of viral vaccine intermediate purification samples. This standard, termed the HCP-PROFILER standard, is composed of a water-soluble bead (READYBEADS technology) which, after being added into the sample, releases unlabeled peptides in controlled amounts. The standard meets three desired criteria: (1) it is composed of multiple peptides, at different concentration levels, allowing construction of a calibration curve covering the dynamic range of HCPs present in the target sample, ensuring quantification accuracy; (2) it demonstrates high batch-to-batch reproducibility, ensuring quantification robustness and consistency over time; and (3) it is easy to use and avoids user-induced analytical biases. In this study, we present the use of the HCP-PROFILER standard for vaccine batches comparison and downstream process performance studies.

Mass spectrometry-based workflow for accurate quantification of Escherichia coli enzymes: how proteomics can play a key role in metabolic engineering.

Metabolic engineering aims to design high performance microbial strains producing compounds of interest. This requires systems-level understanding; genome-scale models have therefore been developed to predict metabolic fluxes. However, multi-omics data including genomics, transcriptomics, fluxomics, and proteomics may be required to model the metabolism of potential cell factories. Recent technological advances to quantitative proteomics have made mass spectrometry-based quantitative assays an interesting alternative to more traditional immuno-affinity based approaches. This has improved specificity and multiplexing capabilities. In this study, we developed a quantification workflow to analyze enzymes involved in central metabolism in Escherichia coli (E. coli). This workflow combined full-length isotopically labeled standards with selected reaction monitoring analysis. First, full-length (15)N labeled standards were produced and calibrated to ensure accurate measurements. Liquid chromatography conditions were then optimized for reproducibility and multiplexing capabilities over a single 30-min liquid chromatography-MS analysis. This workflow was used to accurately quantify 22 enzymes involved in E. coli central metabolism in a wild-type reference strain and two derived strains, optimized for higher NADPH production. In combination with measurements of metabolic fluxes, proteomics data can be used to assess different levels of regulation, in particular enzyme abundance and catalytic rate. This provides information that can be used to design specific strains used in biotechnology. In addition, accurate measurement of absolute enzyme concentrations is key to the development of predictive kinetic models in the context of metabolic engineering.

Accurate quantification of cardiovascular biomarkers in serum using Protein Standard Absolute Quantification (PSAQ™) and selected reaction monitoring.

Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies.

A Validated LC-MS/MS Method for Performing Belatacept Drug Monitoring in Renal Transplantation.

Belatacept, a CTLA4-Ig, was designed to prevent rejection and graft loss in kidney transplant recipients. This immunotherapy showed a long-term clinical benefit mainly on renal function and better glycemic control but was also associated with a higher number of severe infectious diseases, particularly CMV disease, and lymphoproliferative disease. Therapeutic drug monitoring usually guides the benefit-risk assessment of long-term immunosuppression. In this study, an analytical method by LC-MS/MS was developed in 20 microL of plasma for the belatacept quantification. Intra- and inter-assay precision and accuracy were lower than 20% for the limit of quantification, and 15% for higher concentrations. The method was implemented in our lab and provided data about the inter-variability (N = 108) and intra-variability (N = 33) of belatacept concentrations in kidney transplant recipients with a stable renal function, after conversion from a CNI- to a belatacept-based regimen.

Mass spectrometry-based absolute protein quantification: PSAQ™ strategy makes use of "noncanonical" proteotypic peptides.

Absolute quantification of proteins using isotope dilution mass spectrometry requires the selection of proteotypic peptides. When choosing these peptides, a certain number of rules must be respected. Several of these were established to safeguard against quantification errors resulting from the isotopically labeled standard peptides not behaving in the same way as the peptides to be quantified. Of all absolute quantification methods using isotope dilution, Protein Standard for Absolute Quantification (PSAQ(TM) ) offers the maximal protein sequence coverage. In the present study, we show that the PSAQ method presents a previously unreported advantage for protein quantification as it makes use of Met/Cys-containing peptides and peptides-containing miscleavages in addition to proteotypic peptides. By increasing the total number of peptides that can be considered, robustness of quantification is improved, paving the way for a facilitated quantification of low abundant and/or low-molecular-weight proteins.

Complementary use of mass spectrometry and cryo-electron microscopy to assess the maturity of live attenuated dengue vaccine viruses.

Dengue virus (DENV) infection is a global health threat with the potential to affect at least 3.6 billion people living in areas of risk. No specific curative treatments against dengue disease are available and vaccines are currently the only way to prevent the disease. The tetravalent dengue vaccine developed by Sanofi Pasteur has demonstrated significant efficacy in phase III studies and is now licensed in several countries for the prevention of disease in dengue-seropositives over 9 years of age. The vaccine is composed of four recombinant, live, attenuated vaccines (CYD 1-4) based on a yellow fever vaccine 17D (YFV 17D) backbone, each expressing the pre-membrane (prM) and envelope (E) genes of one of the four DENV serotypes. Virus maturity could impact the biological activity of the vaccine viruses. To address this question, the maturity of the four vaccine viruses used in phase III clinical studies was assessed by two complementary techniques: mass spectrometry (MS) and cryo-electron microscopy (cryoEM). MS assessed viral maturity at the molecular level by quantifying specifically the prM, and M proteins. CryoEM provided information at the particle level, allowing visualizing the different phenotypes of viral particles: spiky (immature), smooth/bumpy (mature), and mixed (partially mature). Results of the two assays used in this study show that all four CYD dengue vaccine viruses present in lots used in phase III efficacy trials, display in the majority a mature phenotype.

Noumeavirus replication relies on a transient remote control of the host nucleus.

Acanthamoeba are infected by a remarkable diversity of large dsDNA viruses, the infectious cycles of which have been characterized using genomics, transcriptomics and electron microscopy. Given their gene content and the persistence of the host nucleus throughout their infectious cycle, the Marseilleviridae were initially assumed to fully replicate in the cytoplasm. Unexpectedly, we find that their virions do not incorporate the virus-encoded transcription machinery, making their replication nucleus-dependent. However, instead of delivering their DNA to the nucleus, the Marseilleviridae initiate their replication by transiently recruiting the nuclear transcription machinery to their cytoplasmic viral factory. The nucleus recovers its integrity after becoming leaky at an early stage. This work highlights the importance of virion proteomic analyses to complement genome sequencing in the elucidation of the replication scheme and evolution of large dsDNA viruses.

Development of a Protein Standard Absolute Quantification (PSAQ™) assay for the quantification of Staphylococcus aureus enterotoxin A in serum.

Enterotoxin A (SEA) is a staphylococcal virulence factor which is suspected to worsen septic shock prognosis. However, the presence of SEA in the blood of sepsis patients has never been demonstrated. We have developed a mass spectrometry-based assay for the targeted and absolute quantification of SEA in serum. To enhance sensitivity and specificity, we combined an immunoaffinity-based sample preparation with mass spectrometry analysis in the selected reaction monitoring (SRM) mode. Absolute quantification of SEA was performed using the PSAQ™ method (Protein Standard Absolute Quantification), which uses a full-length isotope-labeled SEA as internal standard. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were estimated at 352pg/mL and 1057pg/mL, respectively. SEA recovery after immunocapture was determined to be 7.8±1.4%. Therefore, we assumed that less than 1femtomole of each SEA proteotypic peptide was injected on the liquid chromatography column before SRM analysis. From a 6-point titration experiment, quantification accuracy was determined to be 77% and precision at LLOQ was lower than 5%. With this sensitive PSAQ-SRM assay, we expect to contribute to decipher the pathophysiological role of SEA in severe sepsis. This article is part of a Special Issue entitled: Proteomics: The clinical link.