Publications

@article{pmid22308409,

title = {Global kinetic analysis of proteolysis via quantitative targeted proteomics},

author = {Nicholas J Agard and Sami Mahrus and Jonathan C Trinidad and Aenoch Lynn and Alma L Burlingame and James A Wells},

doi = {10.1073/pnas.1117158109},

issn = {1091-6490},

year  = {2012},

date = {2012-02-01},

urldate = {2012-02-01},

journal = {Proc Natl Acad Sci U S A},

volume = {109},

number = {6},

pages = {1913--1918},

abstract = {Mass spectrometry-based proteomics is a powerful tool for identifying hundreds to thousands of posttranslational modifications in complex mixtures. However, it remains enormously challenging to simultaneously assess the intrinsic catalytic efficiencies (k(cat)/K(M)) of these modifications in the context of their natural interactors. Such fundamental enzymological constants are key to determining substrate specificity and for establishing the timing and importance of cellular signaling. Here, we report the use of selected reaction monitoring (SRM) for tracking proteolysis induced by human apoptotic caspases-3, -7, -8, and -9 in lysates and living cells. By following the appearance of the cleaved peptides in lysate as a function of time, we were able to determine hundreds of catalytic efficiencies in parallel. Remarkably, we find the rates of substrate hydrolysis for individual caspases vary greater than 500-fold indicating a sequential process. Moreover, the rank-order of substrate cutting is similar in apoptotic cells, suggesting that cellular structures do not dramatically alter substrate accessibility. Comparisons of extrinsic (TRAIL) and intrinsic (staurosporine) inducers of apoptosis revealed similar substrate profiles, suggesting the final proteolytic demolitions proceed by similarly ordered plans. Certain biological processes were rapidly targeted by the caspases, including multiple components of the endocyotic pathway and miRNA processing machinery. We believe this massively parallel and quantitative label-free approach to obtaining basic enzymological constants will facilitate the study of proteolysis and other posttranslational modifications in complex mixtures.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22175275,

title = {Substance P derivatives as versatile tools for specific delivery of various types of biomolecular cargo},

author = {Shahir S Rizk and Agnieszka Misiura and Marcin Paduch and Anthony A Kossiakoff},

doi = {10.1021/bc200496e},

issn = {1520-4812},

year  = {2012},

date = {2012-01-01},

urldate = {2012-01-01},

journal = {Bioconjug Chem},

volume = {23},

number = {1},

pages = {42--46},

abstract = {The use of proteins or nucleic acids as therapeutic agents has been severely hampered by their intrinsic inability to cross the cell membrane. Moreover, common techniques for driving the delivery of macromolecules lack the ability to distinguish between healthy and diseased tissue, precluding their clinical use. Recently, receptor-mediated delivery (RMD) has emerged as a technology with the potential to circumvent the obstacles associated with the delivery of drug targets by utilizing the natural endocytosis of a ligand upon binding to its receptor. Here, we describe the synthesis of variants of substance P (SP), an eleven amino acid neuropeptide ligand of the neurokinin type 1 receptor (NK1R), for the delivery of various types of cargo. The variants of SP were synthesized with an N-terminal maleimide moiety that allows conjugation to surface thiols, resulting in a nonreducible thioether. Cargos lacking an available thiol are conjugated to SP using commercially available cross-linkers. In addition to the delivery of proteins, we expand the use of SP to include nuclear delivery of DNA fragments that are actively expressed in the target cells. We also show that SP can be used to deliver whole bacteriophage particles as well as polystyrene beads up to 1 μm in diameter. The results show the ability of SP to deliver cargo of various sizes and chemical properties that retain their function within the cell. Furthermore, the overexpression of the NK1R in many tumors provides the potential for developing targeted delivery reagents that are specific toward diseased tissue.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}