Attribute Analytics Performance Metrics from the MAM Consortium Interlaboratory Study.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms.

New Peak Detection Performance Metrics from the MAM Consortium Interlaboratory Study.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671.

Network integration and modelling of dynamic drug responses at multi-omics levels.

Uncovering cellular responses from heterogeneous genomic data is crucial for molecular medicine in particular for drug safety. This can be realized by integrating the molecular activities in networks of interacting proteins. As proof-of-concept we challenge network modeling with time-resolved proteome, transcriptome and methylome measurements in iPSC-derived human 3D cardiac microtissues to elucidate adverse mechanisms of anthracycline cardiotoxicity measured with four different drugs (doxorubicin, epirubicin, idarubicin and daunorubicin).

Investigation on Sugar-Protein Connectivity in Salmonella O-Antigen Glycoconjugate Vaccines.

Invasive nontyphoidal Salmonella disease, for which licensed vaccines are not available, is a leading cause of bloodstream infections in Africa. The O-antigen portion of lipopolysaccharide is a good target for protective immunity. Covalent conjugation of the O-antigen to a carrier protein increases its immunogenicity and O-antigen based glycoconjugate vaccines are currently under investigation at the preclinical stage.

Endothelial cell adhesion to the extracellular matrix induces c-Src-dependent VEGFR-3 phosphorylation without the activation of the receptor intrinsic kinase activity.

Rationale: Integrins cooperate with growth factor receptors to promote downstream signaling for cell proliferation and migration. However, the mechanism of receptor activation is still unknown.

Objective: To analyze the mechanism of phosphorylation of the vascular endothelial growth factor receptor (VEGFR)-3 by cell adhesion.

Robust MS quantification method for phospho-peptides using 18O/16O labeling.

Background: Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using 18O/16O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-beta treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable.

Increased expression of the oligopeptidase THOP1 is a neuroprotective response to Abeta toxicity.

In a comprehensive proteomics study aiming at the identification of proteins associated with amyloid-beta (Abeta)-mediated toxicity in cultured cortical neurons, we have identified Thimet oligopeptidase (THOP1). Functional modulation of THOP1 levels in primary cortical neurons demonstrated that its overexpression was neuroprotective against Abeta toxicity, while RNAi knockdown made neurons more vulnerable to amyloid peptide. In the TgCRND8 transgenic mouse model of amyloid plaque deposition, an age-dependent increase of THOP1 expression was found in brain tissue, where it co-localized with Abeta plaques.

Sequential detergent fractionation of primary neurons for proteomics studies.

Proteomics studies employing primary neurons are difficult due to the neurons' characteristics. We have developed a detergent-based fractionation method which reduces complexity of the protein extracts, is sufficiently fast to allow differential proteomics analysis after treatments of neurons for short time periods, can be applied to small numbers of cells directly in culture plates, and allows differential extraction of proteins in a compartment-specific manner. The sequential use of detergent-containing buffers on neurons in culture plates yields four extracts enriched in cytosolic, membrane-bound or enclosed, nuclear, and cytoskeletal proteins.

Detection of phosphorylation patterns in rat cortical neurons by combining phosphatase treatment and DIGE technology.

Although protein phosphorylation is probably the most studied post-translational modification occurring in cells, the number of proteins, which are the target of this modification, is still largely unknown. Increasing the coverage of the phosphoproteome as well as the detection of variation at the phosphorylation level would be very helpful for understanding the mechanisms of cell life and the modifications of the cell state leading to pathological conditions such as neurodegeneration. In order to further investigate variations occurring at the phosphorylation level, we have initiated the creation of a reference map of phosphorylated proteins in rat cortical neurons, employing a combination of phosphatase treatment and 2-DE/differential in gel electrophoresis technology.

Phosphoproteome analysis.

Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult task especially if quantitative comparisons have to be made.