Quantification of in vivo site-specific Asp isomerization and Asn deamidation of mAbs in animal serum using IP-LC-MS.

BACKGROUND: Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern if located within the complementarity-determining region of therapeutic antibodies. Questions arise as to the extent of modification occurring in circulation due to potential exposure of the therapeutic antibody to different pH regimes. RESULTS: To enable evaluation of site-specific isomerization and deamidation of human mAbs in vivo, immunoprecipitation (IP) has been combined with LC-MS providing selective enrichment, separation and detection of naive and modified forms of tryptic peptides comprising complementarity-determining region sequences. CONCLUSION: IP-LC-MS can be applied to simultaneously quantify in vivo drug concentrations and measure the extent of isomerization or deamidation in PK studies conducted during the drug discovery stage.

Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry.

Quantitation of human dystrophin protein in muscle biopsies is a clinically relevant endpoint for both diagnosis and response to dystrophin-replacement therapies for dystrophinopathies. A robust and accurate assay would enable the use of dystrophin as a surrogate biomarker, particularly in exploratory Phase 2 trials. Currently available methods to quantitate dystrophin rely on immunoblot or immunohistochemistry methods that are not considered robust. Here we present a mass spectrometry based approach to accurately quantitate dystrophin protein in a total protein extract from human muscle biopsies. Our approach uses a combination of stable isotope labeled dystrophin as a spike-in standard, gel electrophoresis and high precision mass spectrometry to detect and quantitate multiple peptides of dystrophin within a complex protein mixture. The method was found highly reproducible and linear over a wide dynamic range, detecting as low as 5% of dystrophin relative to the normal amount in healthy individuals.

The balance between pro-apoptotic and anti-apoptotic pathways in the failing myocardium.

The purpose of this review is to highlight those circulating molecules, membrane receptors, and signaling pathways that initiate, potentiate, or conversely, inhibit apoptosis within cardiomyocytes. This review focuses on pathways directly related to the failing heart and discusses the limitations of current methodologies for assessing cardiomyocellular apoptosis. It is important to note that the adrenergic, reactive oxygen species, and proinflammatory cytokine signaling pathways are not the only pro-apoptotic pathways active in the myocardium, nor are IL-6-related cytokine, calcineurin, and IGF-1/PI3K/Akt signaling pathways the only anti-apoptotic pathways active in the myocardium. However, they are among the best-characterized apoptosis-mediating pathways and therefore they may serve as foundation for future studies aimed at identifying novel apoptotic regulating pathways active in cardiomyocytes. Considering the short history of studying cardiomyocellular apoptosis, a tremendous body of knowledge has been collected. Understandably, much more work remains. Tomorrow's studies must (1) continue to examine the signaling pathways mediating cardiomyocellular apoptosis by focusing on the links to the ubiquitous apoptosis effectors, (2) use the expanding body of knowledge to develop more specific inhibitors of apoptosis, and then (3) confirm the causal relationship of cardiomyocellular apoptosis and cardiac dysfunction in physiologic models of cardiac challenge.