Rapid Highly-Efficient Digestion and Peptide Mapping of Adeno-Associated Viruses.

Adeno-associated viruses (AAVs) comprise an area of rapidly growing interest due to their ability to act as a gene delivery vehicle in novel gene therapy strategies and vaccine development. Peptide mapping is a common technique in the biopharmaceutical industry to confirm the correct sequence, product purity, post-translational modifications (PTMs), and stability. However, conventional peptide mapping is time-consuming and has proven difficult to reproduce with viral capsids because of their high structural stability and the suboptimal localization of trypsin cleavage sites in the AAV protein sequences. In this study, we present an optimized peptide mapping-based workflow that provides thorough characterization within 1 day. This workflow is also highly reproducible due to its simplicity having very few steps and is easy to perform proteolytic digestion utilizing thermally stable pepsin, which is active at 70 °C in acidic conditions. The acidic conditions of the peptic digestions drive viral capsid denaturation and improve cleavage site accessibility. We characterized the efficiency and ease of digestion through peptide mapping of the AAV2 viral capsid protein. Using nanoflow liquid chromatography coupled with tandem mass spectrometry, we achieved 100% sequence coverage of the low-abundance VP1 capsid protein with a digestion process taking only 10 min to prepare and 45 min to complete the digestion.

Affinity classification of kinase inhibitors by mass spectrometric methods and validation using standard IC(50) measurements.

Protein kinases have emerged as a major drug target in the last years. Since more than 500 kinases are encoded in the human genome, cross-reactivity of a majority of kinase inhibitors causes problems. Tools are required for a rapid classification of inhibitors according to their affinity for a certain target to refine the search for new, more specific lead compounds. Mass spectrometry (MS) is increasingly used in pharmaceutical research and drug discovery to investigate protein-ligand interactions and determination of binding affinities. We present a comparison of different existing nanoelectrospray-MS based methods to quantify binding affinities and qualitatively rank, by competitive experiments, the affinity of several clinical inhibitors. We also present a new competitive method which is derived from our previous work for quantitative assessment of binding strengths (Wortmann et al., J. Mass Spectrom. 2008, 43(5), 600-608). The human kinases studied for this purpose were p38alpha (MAPK14) and LCK (lymphocyte specific kinase), and their interaction with 17 known small molecule kinase inhibitors was probed. Moreover, we present a new method to differentiate type I from type II inhibitors (Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2(7), 358-364) based on a kinetic experiment with direct MS read-out of the noncovalent complex between the human kinase and the inhibitor. This method was successfully applied to p38alpha binding to BIRB796, as well as to a BIRB796 analogue. Quantitative determination of the binding strength is also described. The results of our competitive experiments for the affinity classification of different inhibitors, as well as the results for the kinetic study, are in good agreement with IC(50) measurements and data found in the literature.