Quantitation of low abundant soluble biomarkers using high sensitivity Single Molecule Counting technology.

Quantitation of biomarkers in biofluids plays a central role in basic research to management of patient care and is routinely used in clinical laboratories and academic institutions. Standard immunoassays, such as an enzyme-linked immunosorbent assay (ELISA), have provided understanding of both normal and pathological processes for many decades. However, in more recent decades, new immunoassay technologies have uncovered numerous analytes in blood that were once undetectable using traditional ELISAs.

Rapid online proteolytic mapping of PEGylated rhGH for identity confirmation, quantitation of methionine oxidation and quantitation of UnPEGylated N-terminus using HPLC with UV detection.

Proteolytic mapping is a widely used tool in the BioPharmaceutical Industry for the analysis of post-translation modifications as well as confirmation of protein identity by comparison to a well-characterized reference standard. This manuscript presents an integrated chromatographic approach which provides the ability to rapidly digest and analyze a PEGylated rhGH for methionine oxidation, identity confirmation and free (unPEGylated) N-terminal peptide by RP-HPLC using UV detection at 280 nm. This approach utilizes an online procedure in which the digestion step is integrated to the RP-HPLC analysis via an external column switching valve.

Structural bioinformatics-based prediction of exceptional selectivity of p38 MAP kinase inhibitor PH-797804.

PH-797804 is a diarylpyridinone inhibitor of p38alpha mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38alpha inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38alpha kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180 degrees rotation of the Met109-Gly110 peptide bond.