Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.

Polyglycidols are flexible hydrophilic polyethers that are potentially biocompatible polymers based on their similarities to the well-studied poly(ethyleneglycol). Polyglycidols can be prepared as branched or linear polymers by suitable synthetic methods. Biocompatibility testing of these polymers conducted in vitro as well as in vivo are reported here. The in vitro studies included hemocompatibility testing for effects on coagulation (PT and APTT), complement activation, red blood cell aggregation, and whole blood viscosity measurements. In vitro cytotoxicity experiments were also conducted. The results were compared with some of the common biocompatible polymers already in human use. Results from these studies show that polyglycidols are highly biocompatible. Hyperbranched polyglycidols were found to be well tolerated by mice even when injected in high doses.

Very high molecular weight (M n up to 700 000) and narrowly polydispersed (PDI = 1.1−1.4) hyperbranched polyglycerols (HPG) were synthesized by ring-opening multibranching polymerization of glycidol using dioxane as an emulsifying agent. Broader molecular weight distributions with low molecular weight fractions were obtained when diglyme was used as the emulsifying agent. But the low molecular weight fractions could be removed by dialysis. Isolated yields in both the cases were 70−90%. The different result in the case of dioxane may be due to faster cation exchange which leads to low polydispersites. HPGs of various molecular weights were characterized by a GPC system coupled with a multiangle laser light scattering detector and a triple detector array. The intrinsic viscosities were low for these polymers and did not increase with molecular weight. The dimensions of these polymers (R g, R h, R η) and their dependence on molecular weights are described. The hydrodynamic radii were very small with dimensions similar to those of dendrimers. Our results show that these polymers are very compact and have spherical conformations in water with no indications of aggregate formation. The melt viscoelastic properties were also studied. Despite their self-similar structures, depending on the type of solvent used to synthesize them (diglyme vs dioxane), topologically restricted configurations are produced that result in completely different entanglement dynamics.

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Phospholipids in the cell membranes of all eukaryotic cells contain phosphatidyl choline (PC) as the headgroup. Here we show that hyperbranched polyglycerols (HPGs) decorated with the 'PC-inverse' choline phosphate (CP) in a polyvalent fashion can electrostatically bind to a variety of cell membranes and to PC-containing liposomes, the binding strength depending on the number density of CP groups per macromolecule. We also show that HPG-CPs can cause cells to adhere with varying affinity to other cells, and that binding can be reversed by subsequent exposure to low molecular weight HPGs carrying small numbers of PCs. Moreover, PC-rich membranes adsorb and rapidly internalize fluorescent HPG-CP but not HPG-PC molecules, which suggests that HPG-CPs could be used as drug-delivery agents. CP-decorated polymers should find broad use, for instance as tissue sealants and in the self-assembly of lipid nanostructures.